Up-converting reporters for biological and other assays

ABSTRACT

The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08962,673filed Nov. 3, 1997, now abandoned which is a continuation-in-part ofapplication Ser. No. 08/482,203 filed Jun. 7, 1995, now U.S. Pat. No.5,698,397 which is a division of application Ser. No. 08/416,023 filedMar. 30, 1995, now U.S. Pat. No. 5,674,698, which is acontinuation-in-part of application Ser. No. 08/381,006 (now abandoned),filed Jan. 30, 1995, which is a continuation of Ser. No. 07/946,068filed Sep. 14, 1992 (now abandoned). This application also claimspriority benefits to provisional application Ser. No. 60/037,392 filedFeb. 7, 1997. Each of these applications is specifically incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The invention relates generally to detectable labels and compositionsuseful in assay methods for detecting soluble, suspended, or particulatesubstances or analytes such as proteins, carbohydrates, nucleic acids,bacteria, viruses, and eukaryotic cells and more specifically relates tocompositions and methods that include luminescent (phosphorescent orfluorescent) labels.

Methods for detecting specific macromolecular species, such as proteins,drugs, and polynucleotides, have proven to be very valuable analyticaltechniques in biology and medicine, particularly for characterizing themolecular composition of normal and abnormal tissue samples and geneticmaterial. Many different types of such detection methods are widely usedin biomedical research and clinical laboratory medicine. Examples ofsuch detection methods inclide: immunoassays, immunochemical stainingfor microscopy, fluorescence-activated cell sorting (FACS), nucleic acidhybridization, water sampling, air sampling, and others.

Typically, a detection method employs at least one analytical reagentthat binds to a specific target macromolecular species and produces adetectable signal. These analytical reagents typically have twocomponents: (1) a probe macromolecule, for example, an antibody oroligonucleotide, that can bind a target macromolecule with a high degreeof specificity and affinity, and (2) a detectable label, such as aradioisotope or covalently-linked fluorescent dye molecule. In general,the binding properties of the probe macromolecule define the specificityof the detection method, and the detectability of the associated labeldetermines the sensitivity of the detection method. The sensitivity ofdetection is in turn related to both the type of label employed and thequality and type of equipment available to detect it.

For example, radioimmunoassays (RIA) have been among the most sensitiveand specific analytical methods used for detecting and quantitatingbiological macromolecules. Radioimmunoassay techniques have been used todetect and measure minute quantities of specific analytes, such aspolypeptides, drugs, steroid hormones, polynucleotides, metabolites, andtumor markers, in biological samples. Radioimmunoassay methods employimmunoglobulins labeled with one or more radioisotopes as the analyticalreagent. Radiation (alpha, beta, or gamma) produced by decay of theattached radioisotope label serves as the signal which can be detectedand quantitated by various radiometric methods.

Radioisotopic labels possess several advantages, such as: very highsensitivity of detection, very low background signal, and accuratemeasurement with precision radiometric instruments (scintillation andgamma counters) or with inexpensive and sensitive autoradiographictechniques. However, radioisotopic labels also have several.disadvantages, such as: potential health hazards, difficulty indisposal, special licensing requirements, and instability (radioactivedecay and radiolysis). Further, the fact that radioisotopic labelstypically do not produce a strong (i.e., non-Cerenkov) signal in theultraviolet, infrared, or visible portions of the electromagneticspectrum makes radioisotopes generally unsuitable as labels forapplications, such as microscopy, image spectroscopy, and flowcytometry, that employ optical methods for detection.

For these and other reasons, the fields of clinical chemistry, water andair monitoring, and biomedical research have sought alternativedetectable labels that do not require radioisotopes. Examples of suchnon-radioactive labels include: (1) enzymes that catalyze conversion ofa chromogenic substrate to an insoluble, colored product (e.g., alkalinephosphatase, beta-galactosidase, horseradish peroxidase) or catalyze areaction that yields a fluorescent or luminescent product (e.g.,luciferase) (Beck and Koster (1990) Anal. Chem. 62:2258; Durrant, I.(1990) Nature 346: 297; Analytical Applications of Bioluminescence andChemiluminescence (1984) Kricka et al. (Eds.) Academic Press, London),and (2) direct fluorescent labels (e.g., fluorescein isothiocyanate,rhodamine, Cascade blue), which absorb electromagnetic energy in aparticular absorption wavelength spectrum and subsequently emit visiblelight at one or more longer (i.e., less energetic) wavelengths.

Using enzymes and phosphorescent/fluorescent or colorimetric detectablelabels offers the significant advantage of signal amplification, since asingle enzyme molecule typically has a persistent capacity to catalyzethe transformation of a chromogenic substrate into detectable product.With appropriate reaction conditions and incubation time, a singleenzyme molecule can produce a large amount of product, and hence yieldconsiderable signal amplification. However, detection methods thatemploy enzymes as labels disadvantageously require additional proceduresand reagents in order to provide a proper concentration of substrateunder conditions suitable for the production and detection of thecolored product. Further, detection methods that rely on enzyme labelstypically require prolonged time intervals for generating detectablequantities of product, and also generate an insoluble product that isnot attached to the probe molecule.

An additional disadvantage of enzyme labels is the difficulty ofdetecting multiple target species with enzyme-labeled probes. It isproblematic to optimize reaction conditions and development time(s) fortwo or more discrete enzyme label species and, moreover, there is oftenconsiderable spectral overlap in the chromophore end products whichmakes discrimination of the reaction products difficult.

Fluorescent labels do not offer the signal amplification advantage ofenzyme labels, nonetheless, fluorescent labels possess significantadvantages which have resulted in their widespread adoption inimmunocytochemistry. Fluorescent labels typically are small organic dyemolecules, such as fluorescein, Texas Red, or rhodamine, which can bereadily conjugated to probe molecules, such as immunoglobulins or Staph.aureus Protein A. The fluorescent molecules (fluorophores) can bedetected by illumination with light of an appropriate excitationfrequency and the resultant spectral emissions can be detected byelectro-optical sensors or light microscopy.

A wide variety of fluorescent dyes are available and offer a selectionof excitation and emission spectra. It is possible to selectfluorophores having emission spectra that are sufficiently different soas to permit multitarget detection and discrimination with multipleprobes, wherein each probe species is linked to a different fluorophore.Because the spectra of fluorophores can be discriminated on the basis ofboth narrow band excitation and selective detection of emission spectra,two or more distinct target species can be detected and resolved (Tituset al. (1982) J. Immunol. Methods 50: 193; Nederlof et al. (1989)Cytometry 10: 20; Ploem, J. S. (1971) Ann. NY Acad. Sci. 177:414).

Unfortunately, detection methods which employ fluorescent labels are oflimited sensitivity for a variety of reasons. First, with conventionalfluorophores it is difficult to discriminate specific fluorescentsignals from nonspecific background signals. Most common fluorophoresare aromatic organic molecules which have broad absorption and emissionspectra, with the emission maximum red-shifted 50-100 nm to a longerwavelength than the excitation (i.e., absorption) wavelength. Typically,both the absorption and emission bands are located in the UV/visibleportion of the spectrum. Further, the lifetime of the fluorescenceemission is usually short, on the order of 1 to 100 ns. Unfortunately,these general characteristics of organic dye fluorescence are alsoapplicable to background signals which are contributed by other reagents(e.g., fixative or serum), or autofluorescence or the sample itself(Jongkind et al. (1982) Exp. Cell Res. 138: 409; Aubin, J. E. (1979) J.Histochem. Cytochem. 27: 36). Autofluorescence of optical lenses andreflected excitation light are additional sources of background noise inthe visible spectrum (Beverloo et al. (1991) Cytometry 11: 784; Beverlooet al. (1992) Cytometry 13: 561). Therefore, the limit of detection ofspecific fluorescent signal from typical fluorophores is limited by thesignificant background noise contributed by nonspecific fluorescence andreflected excitation light.

A second problem of organic dye fluorophores that limits sensitivity isphotolytic decomposition of the dye molecule (i.e., photobleaching).Thus, even in situations where background noise is relatively low, it isoften not possible to integrate a weak fluorescent signal over a longdetection time, since the dye molecules decompose as a function ofincident irradiation in the UV and near-UV bands.

However, because fluorescent labels are attractive for variousapplications, several alternative fluorophores having advantageousproperties for sensitive detection have been proposed. One approach hasbeen to employ organic dyes comprising a phycobiliprotein acceptormolecule dye that emits in the far red or near infrared region of thespectrum where nonspecific fluorescent noise is reduced.Phycobiliproteins are used in conjunction with accessory molecules thateffect a large Stokes shift via energy transfer mechanisms (U.S. Pat.No. 4,666,862; Oi et al. (1982) J. Cell. Biol. 93: 891).Phycobiliprotein labels reduce the degree of spectral overlap betweenexcitation frequencies and emission frequencies. An alternative approachhas been to use cyanine dyes which absorb in the yellow or red regionand emit in the red or far red where autofluorescence is reduced(Mujumbar et al. (1989) Cytometry 10: 11).

However, with both the phycobiliproteins and the cyanine dyes theemission frequencies are red-shifted (i.e., frequency downshifted) andemission lifetimes are short, therefore background autofluorescence isnot completely eliminated as a noise source. More importantly perhaps,phycobiliproteins and cyanine dyes possess several distinctdisadvantages: (1) emission in the red, far red, and near infraredregion is not well-suited for detection by the human eye, hampering theuse of phycobiliprotein and cyanine labels in optical fluorescencemicroscopy, (2) cyanines, phycobiliproteins, and the coupled accessorymolecules (e.g., Azure A) are organic molecules susceptible tophotobleaching and undergoing undesirable chemical interactions withother reagents, and (3) emitted radiation is down-converted, i.e., oflonger wavelength(s) than the absorbed excitation radiation. Forexample, Azure A absorbs at 632 nm and emits at 645 nm, andallophycocyanin absorbs at 645 nm and emits at 655 nm, and thereforeautofluorescence and background noise from scattered excitation light isnot eliminated.

Another alternative class of fluorophore that has been proposed are thedown-converting luminescent lanthanide chelates (Soini and Lovgren(1987) CRC Crit. Rev. Anal. Chem. 18: 105; Leif et al. (1977) Clin.Chem. 23: 1492; Soini and Hemmila (1979) Clin. Chem. 25: 353; Seveus etal. (1992) Cytometry 13: 329). Down-converting lanthanide chelates areinorganic phosphors which possess a large downward Stokes shift (i.e.,emission maxima is typically at least 100 nm greater than absorptionmaxima) which aids in the discrimination of signal from scatteredexcitation light. Lanthanide phosphors possess emission lifetimes thatare sufficiently long (i.e., greater than 1 mu s) to permit their use intime-gated detection methods which can reduce, but not totallyeliminate, noise caused by shorter-lived autofluorescence and scatteredexcitation light. Further, lanthanide phosphors possess narrow-bandemission, which facilitates wavelength discrimination against backgroundnoise and scattered excitation light, particularly when a laserexcitation source is utilized (Reichstein et al. (1988) Anal. Chem. 60:1069). Recently, enzyme-amplified lanthanide luminescence usingdown-converting lanthanide chelates has been proposed as a fluorescentlabeling technique (Evangelista et al. (1991) Anal. Biochem. 197: 213;Gudgin-Templeton et al. (1991) Clin Chem. 37:1506).

Until recently, down-converting lanthanide phosphors have had thesignificant disadvantage that their quantum efficiency in aqueous(oxygenated) solutions is so low as to render them unsuitable forcytochemical staining. Beverloo et al. (op.cit.) have described aparticular down-converting lanthanide phosphor (yttrium oxysulfideactivated with europium) that produces a signal in aqueous solutionswhich can be detected by time-resolved methods. Seveus et al. (op.cit.)have used down-converting europium chelates in conjunction withtime-resolved fluorescence microscopy to reject the signal from promptfluorescence and thereby reduce autofluorescence.

However, the down-converting lanthanide phosphor of Beverloo et al. andthe europium chelate of Seveus et al. require excitation wavelengthmaxima that are in the ultraviolet range, and thus produce significantsample autofluorescence and background noise (e.g., serum and/orfixative fluorescence, excitation light scattering and refraction, etc.)that must be rejected (e.g., by fillers or time-gated signal rejection).Further, excitation with ultraviolet irradiation damages nucleic acidsand other biological macromolecules, posing serious problems forimmunocytochemical applications where it is desirable to preserve theviability of living cells and retain cellular structures (e.g., FACS,cyto-architectural microscopy).

Laser scanning fluorescence microscopy has been used for two-photonexcitation of a UV-excitable fluorescent organic dye, Hoechst 33258,using a stream of strongly focused laser pulses (Denk et al. (1990)Science 248: 73). The organic fluorphore used by Denk et al. wassignificantly photobleached by the intense, highly focused laser lightduring the course of imaging. Motsenbocker et al. (EP 476 556) describesa method to increase luminol chemiluminescence by adding a dye catalystthat absorbs long wavelength radiation (deep red light) and subsequentlyreacts with molecular oxygen to generate an oxidant which can itselfreact with luminol and produce oxidized luminol which emits blue light.Gavrilovic (U.S. Pat. No. 5,166,948) discloses a method and apparatusfor optical pumping of infrared pump light to a visible or ultravioletemission light having a wavelength shorter than the pump light (i.e.,up-converted emission).

Thus, there exists a significant need in the art for labels anddetection methods that permit sensitive optical and/or spectroscopicdetection of specific label signal(s) with essentially total rejectionof nonspecific background noise, and which are compatible with intactviable cells and aqueous or airborne environments.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The present invention provides labels, detection methods, and detectionapparatus which permit ultrasensitive detection of cells, biologicalmacromolecules, and other analytes, which can be used for multipletarget detection and target discrimination. The up-converting labels ofthe invention permit essentially total rejection of non-specificbackground autofluorescence and are characterized by excitation andemitted wavelengths that are typically in the infrared or visibleportions of the spectrum, respectively, and thus avoid the potentiallydamaging effects of ultraviolet radiation. The up-converting labels ofthe invention convert long-wavelength excitation radiation (e.g.,near-IR) to emitted radiation, which is generally about one-half toone-third the wavelength of the excitation wavelength. Since backgroundfluorescence in the visible range is negligible if near-IR excitationwavelengths are used, the use of up-converting labels providesessentially background-free detection of signal.

In brief, the invention provides the use of luminescent materials thatare capable of multiphoton excitation and have upshifted emissionspectra. In one embodiment of the invention, up-converting phosphors(i.e., which absorb multiple photons in a low frequency band and emit ina higher frequency band) are used as labels which can be linked to oneor more probes, such as an immunoglobulin, polynucleotide, streptavidin,Protein A, receptor ligand, or other probe molecule. In an anotherembodiment, up-converting organic dyes serve as the label. The organicdye labels and phosphor labels of the invention are highly compatiblewith automated diagnostic testing, microscopic imaging applications, andcoded particle detection, among many other applications.

The nature of the invention provides considerable flexibility in theapparatus for carrying out the methods. As a general matter, theexcitation source may be any convenient light source, includinginexpensive near-infrared laser diodes or light-emitting diodes (LEDs),and the detector may be any convenient detector, such as a photodiode.In the case of a single reporter, the apparatus includes a laser diodecapable of emitting light at one or more wavelengths in the reporter'sexcitation band and a detector that is sensitive to at least somewavelengths in the reporter's emission band. The laser light ispreferably focused to a small region in the sample, and light emanatingfrom that region is collected and directed to the detector. Anelectrical signal representing the intensity of light in the emissionband provides a measure of the amount of reporter present. Depending onthe detector's spectral response, it may be necessary to provide afilter to block the excitation light.

Simultaneous detection of multiple reporters is possible, at least wherethe reporters have different excitation bands or different emissionbands. Where the excitation bands differ, multiple laser diodes emittingat respective appropriate wavelengths are combined using a wavelengthdivision multiplexer or other suitable techniques, such as frequencylabeling, frequency modulation, and lock-in detector device. If theemission bands are different (whether or not the excitation bands aredifferent), light in the different emission bands is separated and sentto multiple detectors. If the emission bands overlap, a single detectormay be used, but other detection techniques are used. One example is touse time multiplexing techniques so that only one reporter is emittingat a given time. Alternatively, the different laser diodes can bemodulated at different characteristic frequencies and lock-in detectionperformed.

Detection methods and detection apparatus of the present inventionenable the ultrasensitive detection of up-converting phosphors andup-converting organic dyes by exploiting what is essentially the totalabsence of background noise (e.g., autofluorescence, serum/fixativefluorescence, excitation light scatter) that are advantageouscharacteristics of up-converting labels. Some embodiments of theinvention utilize time-gated detection and/or wavelength-gated detectionfor optimizing detection sensitivity, discriminating multiple samples,and/or detecting multiple probes on a single sample. Phase-sensitivedetection can also be used to provide discrimination between signal(s)attributable to an up-converting phosphor and background noise (e.g.autofluorescence) which has a different phase shift.

Up-converting organic dyes, such as red-absorbing dyes, also can be usedin an alternate embodiment that converts the photons absorbed by the dyeinto a transient voltage that can be measured using electrodes andconventional electronic circuitry. After having undergone two-photonabsorption the dye is ionized by additional photons from the lightsource (e.g., a laser) leading to short-lived molecular ions whosepresence can be detected and quantified by measuring the transientphotoconductivity following the excitation irradiation. In thisembodiment, resonant multiphoton ionization is used to provide aquantitative measurement of the number and/or concentration of dyemolecules in a sample. Furthermore, essentially all photoions formed inthe irradiated sample contribute to the signal, whereas photons areemitted isotopically and only a fraction can be collected using optics.Measurement of the transient photocurrent effectively transfers theconversion of photons into an electronic signal that is readily measuredwith relatively simple and inexpensive sensors such as electrodes.

In some embodiments, the present invention utilizes one or more opticallaser sources for generating excitation illumination of one or morediscrete frequency(ies). In certain variations of the invention, laserirradiation of an up-converting label can modify the immediate molecularenvironment through laser-induced photochemical processes involvingeither direct absorption or energy transfer; such spatially-controlleddeposition of energy can be used to produce localized damage and/or toprobe the chemical environment of a defined location. In suchembodiments, the up-converting label can preferably act as aphotophysical catalyst.

The invention provides methods for producing targeted damage (e.g.,catalysis) in chemical or biological materials, wherein a probe isemployed to localize a linked up-converting label to a position near atargeted biological structure that is bound by the probe. The localizedup-converting label is excited by one or more excitation wavelengths andemits at a shorter wavelength which may be directly cytotoxic orgenotoxic (e.g., by producing free radicals such as superoxide, and/orby generating thymine-thymine dimers), or which may induce a localphotolytic chemical reaction to produce reactive chemical species in theimmediate vicinity of the label, and hence in the vicinity of thetargeted biological material. Thus, targeting probes labeled with one ormore up-converting labels (e.g., an up-converting inorganic phosphor)may be used to produce targeted damage to biological structures, such ascells, tissues, neoplasms, vasculature, or other anatomical orhistological structures.

Embodiments of the present invention also include up-convertingphosphors which can also be excited by an electron beam or other beam ofenergetic radiation of sufficient energy and are cathodoluminescent.Such electron-stimulated labels afford novel advantages in eliminatingbackground in ultrasensitive biomolecule detection methods. Typically,stimulation of the up-converting phosphor with at least two electrons isemployed to generate a visible-light or UV band emission.

The invention also provides for the simultaneous detection of multipletarget species by exploiting the multiphoton excitation and subsequentbackground-free fluorescence detection of several up-convertingphosphors or up-converting dyes. In one embodiment, severalphosphorsdyes are selected which have overlapping absorption bands whichallow simultaneous excitation at one wavelength (or in a narrowbandwidth), but which vary in emission characteristics such that eachprobe-label species is endowed with a distinguishable fluorescent"fingerprint." By using various methods and devices, the presence andconcentration of each of the phosphors or dyes can be determined.

The invention also provides biochemical assay methods for determiningthe presence and concentration of one or more analytes, typically insolution. The assay methods employ compositions of probes labeled withup-converting phosphors and/or up-converting dyes and apparatus formagnetically and/or optically trapping particles that comprise theanalyte and the labeled probe. In one embodiment, a sandwich assay isperformed, wherein an immobilized probe, immobilized on a particle,binds to a predetermined analyte, producing-an immobilization of thebound analyte on the particle; a second probe, labeled with anup-converting label can then bind to the bound analyte to produce abound sandwich complex containing an up-converting label bound to aparticle. By combining different probe-label combinations, particles ofvarious sizes, colors, and/or shapes with distinct immobilized probe(s),and/or various excitation wavelengths, it is possible to performmultiple assays essentially simultaneously or contemporaneously. Thismultiplex advantage affords detection and quantitation of multipleanalyte species in a single sample. The assay methods are also usefulfor monitoring the progress of a reaction, such as a physical, chemical,biochemical, or immunological reaction, including binding reactions. Forexample, the invention may be used to monitor the progress ofligand-binding reactions, polynucleotide hybridization reactions,including hybridization kinetics and thermodynamic stability ofhybridized polynucleotides.

The invention also provides methods, up-converting labels, andcompositions of labeled binding reagents for performingfluorescence-activated cell sorting (FACS) by flow cytometry usingexcitation radiation that is in the infrared portion of the spectrum anddoes not significantly damage cells. This provides a significantadvantage over present FACS methods which rely on excitationillumination in the ultraviolet portion of the spectrum, includingwavelengths which are known to produce DNA lesions and damage cells.

The invention also provides compositions comprising at least onefluorescent organic dye molecule attached to an inorganic up-convertingphosphor. The fluorescent organic dye molecule is selected from thegroup consisting of: rhodamines, cyanines, xanthenes, acridines,oxazines, porphyrins, and phthalocyanines, and may optionally becomplexed with a heavy metal. The fluorescent organic dye may beadsorbed to the inorganic up-converting phosphor crystal and/or may becovalently attached to a coated inorganic up-converting phosphor, aderivatized vitroceramic up-converting phosphor, or a microencapsulatedinorganic up-converting phosphor. Frequently, covalent conjugationbetween the up-converting inorganic phosphor particles and proteins(e.g., avidin, immunoglobulin) can be accomplished withhetrobifunctional crosslinkers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an optical and electronic block diagram illustratingrepresentative apparatus for performing diagnostics on a sampleaccording to the present invention;

FIG. 2A shows apparatus for implementing phase sensitive detection inthe context of a single channel;

FIG. 2B shows apparatus where first and second laser diodes aremodulated by signals from waveform generators;

FIG. 3 shows apparatus for performing gated detection;

FIG. 4 shows an apparatus for performing diagnostics on a sample usingfirst and second reporters excitation bands centered at lambda 1 andlambda 2, respectively, and having overlapping emission bands nearlambda 3;

FIG. 5A, 5B, 5C show schematically energy state transitions inmulti-photon excitation schemes.

FIG. 6 shows a miniaturized instrument using a hand-held probe;

FIG. 7A shows the use of a charge-coupled device (CCD) array used todetect emissions from a large plurality of binding sites;

FIG. 7B shows the CCD array used in conjunction with a lens array;

FIG. 8 shows an embodiment using optical trapping;

FIG. 9 shows schematically dye coating and encapsulation of anup-converting phosphor particle;

FIG. 10 shows schematically an apparatus for determining particlevelocity and hydrodynamic or aerodynamic properties of a target;

FIG. 11 is a phosphor emission spectrum of sodium yttriumfluoride-ytterbium/erbium up-converting phosphor with an excitationlaser source at a wavelength maximum of 977.2 nm; emission maximum isabout 541.0 nm;

FIG. 12 is an excitation scan of the sodium yttriumfluoride-ytterbium/erbium phosphor excitation spectrum, with emissioncollection window set at 541.0 nm;

FIG. 13 is a time-decay measurement of the phosphor luminescence at541.0 nm after termination of excitation illumination for sodium yttriumfluoride-ytterbium/erbium;

FIG. 14 shows the phosphor emission intensity as a function ofexcitation illumination intensity for a sodium yttriumfluoride-ytterbium/erbium phosphor;

FIG. 15 shows effective single-photon phosphorescence cross-section for0.3 μm particles of Na(Y₀.8 Yb₀.12 Er₀.08)F₄ following excitation with200 Wcm² at 970 nm.

FIG. 16 shows size-dependence of phosphorescence cross-section forNa(Y₀.8 Yb₀.12 Er₀.08)F₄ particles.

FIG. 17A shows a fluorescence scan of an up-converting phosphor reporterin Hepes-buffered saline induced by excitation with a 970-nm lasersource;

FIG. 17B shows a fluorescence spectrum scan of an up-converting phosphorreporter coated with streptavidin in Hepes-buffered saline induced byexcitation with a 970-nm laser source;

FIG. 18A shows an excitation spectrum scan of an up-converting phosphorreporter in Hepes-buffered saline with monochromatic detection ofemission at 541 nm;

FIG. 18B shows an excitation spectrum scan of an up-converting phosphorreporter coated with streptavidin in Hepes-buffered saline withmonochromatic detection of emission at 541 nm;

FIG. 19 shows the integrated signal obtained from samples of(Y₀.86Yb₀.08 Er₀.06)₂ O₂ S showing the relationship between phosphorconcentration and up-converted signal;

FIG. 20 shows schematically one embodiment of an sandwich immunoassayfor detecting an analyte in a solution by binding the analyte (e.g., anantigen target) to a biotinylated antibody and to an immobilizedantibody, wherein the analyte forms a sandwich complex immobilized on asolid substrate superparamagnetic microbead; and

FIG. 21 shows schematically the detection and discrimination of two cellsurface antigens with specific antibodies labeled with two phosphorswith distinct phosphorescence characteristics.

FIG. 22 shows a schematic of an apparatus for phase-sensitive detection.

FIG. 23 show a schematic of a competitive homogeneous assay usingphosphors as labels and fiber optic illumination at a capture surface.

FIG. 24 show a schematic of a competitive homogeneous antigen captureassay using phosphors as labels and a convergent illumination beamfocused on the capture surface.

FIG. 25 shows a schematic of a homogeneous immunoprecipitation assayusing phosphors as labels and a convergent illumination beam focused onthe capture surface wherein the capture surface collectsimmunoprecipitates.

FIG. 26 shows a block diagram of one embodiment of apparatus forcarrying out the present invention on a sample using a microscope.

FIG. 27 is a block diagram of a microtiter plate reader for use with thepresent invention.

FIG. 28 is an illustration of the data for upconverting phosphors inthree test wells.

FIG. 29 is a schematic view of a second embodiment of a hand-held probefor carrying out the present invention.

FIG. 30 illustrates a three channel configuration using interferencefilters.

FIG. 31A is an illustration of an embodiment of the invention in which adiode laser array F1 and a detector array F2 are combined in a singledevice.

FIG. 31B is a detailed view of a small section of the device shown inFIG. 31A.

FIG. 32 is an emission spectrum for up-conversion from neodymiumchelated in EDTA.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, "label" refers to a chemical substituent that produces,under appropriate excitation conditions, a detectable optical signal.The optical signal produced by an excited label is typicallyelectromagnetic radiation in the near-infrared, visible, or ultravioletportions of the spectrum. The labels of the invention are generallyup-converting labels, which means that the chemical substituenttypically absorbs at least two photons at an excitation frequency andsubsequently emits electromagnetic energy at an emission frequencyhigher than the excitation frequency. Thus, there is generally asignificant Stokes shift between the original excitation frequency andthe final emission frequency. A label is generally attached to a probeto serve as a reporter that indicates the presence and/or location ofprobe. The invention encompasses organic and inorganic up-convertinglabels, but preferably employs up-converting inorganic lanthanidephosphors as labels. Thus, a typical label of the invention is asubmicron-size up-converting lanthanide phosphor particle. The label canalternatively comprise a lanthanide ion in a chelate or cage compound.

As used herein, a "probe" refers to a binding component which bindspreferentially to one or more targets (e.g., antigenic epitopes,polynucleotide sequences, macromolecular receptors) with an affinitysufficient to permit discrimination of labeled probe bound to targetfrom nonspecifically bound labeled probe (i.e., background). Generally,the probe-target binding is a non-covalent interaction with a bindingaffinity (K[D]) of at least about 1×10⁶ M-1, preferably with at leastabout 1×10⁷ M⁻¹, and more preferably with an affinity of at least about1×10⁸ M⁻¹ or greater. Antibodies typically have a binding affinity forcognate antigen of about 1×10¹⁰ M⁻¹ or more,. For example but notlimitation, probes of the invention include: antibodies, polypeptidehormones, polynucleotides, streptavidin, Staphlyococcus aureus proteinA, receptor ligands (e.g., steroid or polypeptide hormones), leucinezipper polypeptides, lectins, antigens (polypeptide, carbohydrate,nucleic acid, and hapten epitopes), and others.

As used herein, a "probe-label conjugate" and a "labeled probe" refer toa combination comprising a label attached to a probe. In certainembodiments, more than one label substituent may be attached to a probe.Alternatively, in some embodiments more than one probe may be attachedto a label (e.g., multiple antibody molecules may be attached to asubmicron-size inorganic up-converting phosphor bead). Variousattachment chemistries can be employed to link a label to a probe,including, but not limited to, the formation of: covalent bonds,hydrogen bonds, ionic bonds, electrostatic interactions, and surfacetension (phase boundary) interactions. Attachment of label can alsoinvolve incorporation of the label into or onto microspheres,microparticles, immunobeads, and superparamagnetic magnetic beads(Polysciences, Inc., Warrington, Pa.; Bangs Laboratories, Inc., 979Keystone Way, Carmel, Ind. 46032). For example, inorganic up-convertingphosphor particles can be encapsulated in microspheres that are composedof polymer material that is essentially transparent or translucent inthe wavelength range(s) of the excitation and emitted electromagneticradiation (U.S. Pat. No. 5,132,242, incorporated herein by reference).Such microspheres can be functionalized by surface derivatization withone or more reactive groups (e.g., carboxylate, amino, hydroxylate, orpolyatcrolein) for covalent attachment to a probe, such as a protein.Probe-label conjugates can also comprise a phosphor chelate.

As used herein, the term "target" and "target analyte" refer to theobject(s) that is/are assayed for by the methods of the invention. Forexample but not limitation, targets can comprise polypeptides (e.g.,hGH, insulin, albumin), glycoproteins (e.g., immunoglobulins,thrombomodulin, gamma -glutamyltranspeptidase; Goodspeed et al. (1989)Gene 76: 1), lipoproteins, viruses, microorganisms (e.g., pathogenicbacteria, yeasts), polynucleotides (e.g., cellular genomic DNA, RNA in afixed histological specimen for in situ hybridization, DNA or RNAimmobilized on a nylon or nitrocellulose membrane, viral DNA or RNA in atissue or biological fluid), and pharmaceuticals (i.e., prescribed orover-the-counter drugs listed in the Physicians Drug Reference and/orMerck Manual, or illegal substances such as intoxicants or anabolicsteroids).

As used herein, the term "antibody" refers to a protein consisting ofone or more polypeptides substantially encoded by immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes.Full-length immunoglobulin "light chains" (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes, e.g., gamma (encoding about 330 amino acids). One form ofimmunoglobulin constitutes the basic structural unit of an antibody.This form is a tetramer and consists of two identical pairs ofimmunoglobulin chains, each pair having one light and one heavy chain.In each pair, the light and heavy chain variable regions are togetherresponsible for binding to an antigen, and the constant regions areresponsible for the antibody effector functions. In addition toantibodies, immunoaiobulins may exist in a variety of other formsincluding, for example, Fv, Fab, and F(ab')2, as well as bifunctionalhybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105(1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988)). (See, generally, Hood et al., "Immunology" , Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).Thus, not all immunoglobulins are antibodies. (See, U.S. Ser. No.07/634,278, which is incorporated herein by reference, and Co et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 2869, which is incorporatedherein by reference).

As used herein, "probe polynucleotide" refers to a polynucleotide thatspecifically hybridizes to a predetermined target polynucleotide. Forexample but not limitation, a probe polynucleotide may be a portion of acDNA corresponding to a particular mRNA sequence, a portion of a genomicclone, a synthetic oligonucleotide having sufficient sequence homologyto a known target sequence (e.g., a telomere repeat TTAGGG or an Alurepetitive sequence) for specific hybridization, a transcribed RNA(e.g., from an SP6 cloning vector insert), or a polyamide nucleic acid(Nielsen et al. (1991) Science 254: 1497). Various targetpolynucleotides may be detected by hybridization of a labeled probepolynucleotide to the target sequence(s). For example but notlimitation, target polynucleotides may be: genomic sequences (e.g.,structural genes, chromosomal repeated sequences, regulatory sequences,etc.), RNA (e.g., mRNA, hnRNA, rRNA, etc.), pathogen sequences (e.g.,viral or mycoplasmal DNA or RNA sequences), or transgene sequences.

"Specific hybridization" is defined herein as the formation of hybridsbetween a probe polynucleotide and a target polynucleotide, wherein theprobe polynucleotide preferentially hybridizes to the target DNA suchthat, for example, at least one discrete band can be identified on aSouthern blot of DNA prepared from eukaryotic cells that contain thetarget polynucleotide sequence, and/or a probe polynucleotide in anintact nucleus localizes to a discrete chromosomal locationcharacteristic of a unique or repetitive sequence. In some instances, atarget sequence may be present in more than one target polynucleotidespecies (e.g., a particular target sequence may occur in multiplemembers of a gene family or in a known repetitive sequence). It isevident that optimal hybridization conditions will vary depending uponthe sequence composition and length(s) of the targetingpolynucleotide(s) and target(s), and the experimental method selected bythe practitioner. Various guidelines may be used to select appropriatehybridization conditions (see, Maniatis et al., Molecular Cloning: ALaboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Bergerand Kimmel, Methods in Enzymology, Volume 152 Guide to Molecular CloningTechniques (1987), Academic Press, Inc., San Diego, Calif., Dunn et al.(1989) J. Biol. Chem. 264: 13057 and Goodspeed et al. (1989) Gene 76: 1.

As used herein, the term "label excitation wavelength" refers to anelectromagnetic radiation wavelength that, when absorbed by anup-converting label, produces a detectable fluorescent emission from theup-converting label, wherein the fluorescent emission is of a shorterwavelength (i.e., higher frequency radiation) that the label excitationwavelength. As used herein, the term "label emission wavelength" refersto a wavelength that is emitted from an up-converting label subsequentto, or contemporaneously with, illumination of the up-converting labelwith one or more excitation wavelengths; label emission wavelengths ofup-converting labels are shorter (i.e., higher frequency radiation) thanthe corresponding excitation wavelengths. Both label excitationwavelengths and label emission wavelengths are characteristic toindividual up-converting label species, and are readily determined byperforming simple excitation and emission scans.

Invention Overview

The subject invention encompasses fluorescent labels that are excited byan excitation wavelength and subsequently emit electromagnetic radiationat up-shifted frequencies (i.e., at higher frequencies than theexcitation radiation).

In accordance with the present invention, labels comprisingup-converting inorganic phosphors and/or up-converting organic dyes areprovided for various applications. The up-converting labels of theinvention may be attached to one or more probe(s) to serve as a reporter(i.e., a detectable marker) of the location of the probe(s). Theup-converting labels can be attached to various probes, such asantibodies, streptavidin, protein A, polypeptide ligands of cellularreceptors, polynucleotide probes, drugs, antigens, toxins, and others.Attachment of the up-converting label to the probe can be accomplishedusing various linkage chemistries, depending upon the nature of thespecific probe. For example but not limitation, microcrystallineup-converting lanthanide phosphor particles may be coated with apolycarboxylic acid (e.g., Addition XW 330, Hoechst, Frankfurt, Germany)during milling and various proteins (e.g., immunoglobulin, streptavidinor protein A) can be physically adsorbed to the surface of the phosphorparticle (Beverloo et al. (1991) op.cit., which is incorporated hereinby reference). Alternatively, various inorganic phosphor coatingtechniques can be employed including, but not limited to: spray drying,plasma deposition, and derivatization with functional groups (e.g.,--COOH, --NH₂, --CONH₂) attached by a silane coupling agent to --SiOHmoieties coated on the phosphor particle or incorporated into avitroceramic phosphor particle comprising silicon oxide(s) andup-converting phosphor compositions. Vitroceramic phosphor particles canbe aminated with, for example, aminopropyltriethoxysilane for thepurpose of attaching amino groups to the vitroceramic surface on linkermolecules, however other omega-functionalized silanes can be substitutedto attach alternative functional groups. Probes, such as proteins orpolynucleotides may then be directly attached to the vitroceramicphosphor by covalent linkage, for example through siloxane bonds orthrough carbon-carbon bonds to linker-molecules (e.g., organofunctionalsilylating agents) that are covalently bonded to or adsorbed to thesurface of a phosphor particle. Covalent conjugation between theup-converting inorganic phosphor particles and proteins (e.g., avidin,immunoglobulin) can be accomplished with homobifunctional, or preferablyheterobifunctional, crosslinkers. For example, surface silanization ofthe phosphors with tri(ethoxy)thiopropyl silane leaves a phosphorsurface with a thiol finctionality to which a protein (e.g., antibody)or any compound containing a primary amine can be grafted usingconventional N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) chemistry(Weltman et al. (1983). Other silanization and cross-linking methodscompatible with the inorganic phosphors may be used at the discretion ofthe practitioner.

Microcrystalline up-converting phosphor particles are typically smallerthan about 2 microns in diameter, preferably less than about 1 micron indiameter (i.e., submicron), and more preferably are 0.1 to 0.3 micronsor less in diameter. It is generally most preferred that the phosphorparticles are as small as possible while retaining sufficient quantumconversion efficiency to produce a detectable signal; however, for anyparticular application, the size of the phosphor particle(s) to be usedshould be selected at the discretion of the practitioner. For instance,some applications (e.g., detection of a non-abundant cell surfaceantigen) may require a highly sensitive phosphor label that need not besmall but must have high conversion efficiency and/or absorptioncross-section, while other applications (e.g., detection of an abundantnuclear antigen in a permeablized cell) may require a very smallphosphor particle that can readily diffuse and penetrate subcellularstructures, but which need not have high conversion efficiency.Therefore, the optimal size of inorganic phosphor particle isapplication dependent and is selected by the practitioner on the basisof quantum efficiency data for the various phosphors of the invention.Such conversion efficiency data may be obtained from avaliable sources(e.g., handbooks and published references) or may be obtained bygenerating a standardization curve measuring quantum conversionefficiency as a function of particle size. In some applications, such asthose requiring highly sensitive detection of small phosphor particles,infrared laser diodes are preferably selected as an excitation source.

Although the properties of the up-converting phosphors will be describedin detail in a later section, it is useful to outline the basicmechanisms involved. Up-conversion has been found to occur in certainmaterials containing rare-earth ions in certain crystal materials. Forexample, ytterbium and erbium act as an activator couple in a phosphorhost material such as bariumn-yttrium-fluoride. The ytterbium ions actas the absorber, and transfer energy non-ritdiatively to excite theerbium ions. The emission is thus characteristic of the erbium ion'senergy levels.

Up-Converting Microcrvstalline Phosphors

Although the invention can be practiced with a variety of up-convertinginorganic phosphors, it is believed that the preferred embodiment(s)employ one or more phosphors derived from one of several differentphosphor host materials, each doped with at least one activator couple.Suitable phosphor host materials include: sodium yttrium fluoride(NaYF₄), lanthanum fluoride (LaF₃), lanthanum oxysulfide, yttriumoxysulfide, yttrium fluoride (YF₃), yttrium gallate, yttrium aluminumgarnet, gadolinium fluoride (GdF₃), barium yttrium fluoride (BaYF₅, BaY₂F₈), and gadolinium oxysulfide. Suitable activator couples are selectedfrom: ytterbium/erbium, ytterbium/thulium, and ytterbium/holmium. Otheractivator couples suitable for up-conversion may also be used. Bycombination of these host materials with the activator couples, at leastthree phosphors with at least three different emission spectra (red,green, and blue Nisible light) are provided. Generally, the absorber isytterbium and the emitting center can be selected from: erbium, holmium,terbium, and thulium; however, other up-converting phosphors of theinvention may contain other absorbers and/or emitters. The molar ratioof absorber: emitting center is typically at least about 1:1, moreusually at least about 3:1 to 5:1, preferably at least about 8:1 to10:1, more preferably at least about 11:1 to 20:1, and typically lessthan about 250:1, usually less than about 100:1, and more usually lessthan about 50:1 to 25:1, although various ratios may be selected by thepractitioner on the basis of desired characteristics (e.g., chemicalproperties, manufacturing efficiency, absorption cross-section,excitation and emission wavelengths, quantum efficiency, or otherconsiderations). The ratio(s) chosen will generally also depend upon theparticular absorber-emitter couple(s) selected, and can be calculatedfrom reference values in accordance with the desired characteristics.

The optimum ratio of absorber (e.g., ytterbium) to the emitting center(e.g., erbium, thulium, or holmium) varies, depending upon the specificabsorberemitter couple. For example, the absorber:emitter ratio forYb:Er couples is typically in the range of about 20:1 to about 100:1,whereas the absorber:emitter ratio for Yb:Tm and Yb:Ho couples istypically in the range of about 500:1 to about 2000:1. These differentratios are attributable to the different matching energy levels of theEr, Tm, or Ho with respect to the Yb level in the crystal. For mostapplications, up-converting phosphors may conveniently comprise about10-30% Yb and either: about 1-2% Er, about 0.1-0.05% Ho, or about0.1-0.05% Tm, although other formulations may be employed.

Some embodiments of the invention employ inorganic phosphors that areoptimally excited by infrared radiation of about 950 to 1000 nm,preferably about 960 to 980 nm. For example but not limitation, amicrocrystalline inorganic phosphor of the formula YF₃ :Yb₀.10 Er₀.01exhibits a luminescence intensity maximum at an excitation wavelength ofabout 980 nm. Inorganic phosphors of the invention typically haveemission maxima that are in the visible range. For example, specificactivator couples have characteristic emission spectra: ytterbium-erbiumcouples have emission maxima in the red or green portions of the visiblespectrum, depending upon the phosphor host; ytterbium-holmium couplesgenerally emit maximally in the green portion, ytterbium-thuliumtypically have an emission maximum in the blue range, andytterbiumterbium usually emit maximally in the green range. For example,Y₀.80 Yb₀.19 Er₀.01 F₂ emits maximally in the green portion of thespectrum.

Although up-converting inorganic phosphor crystals of various formulaeare suitable for use in the invention, the following formulae, providedfor example and not to limit the invention, are generally suitable:

Na(Y_(x) Yb_(y) Er_(z))F₄ : x is 0.7 to 0.9, y is 0.09 to 0.29, and z is0.05 to 0.01;

Na(Y_(x) Yb_(y) Ho_(z))F₄ : x is 0.7 to 0.9, y is 0.0995 to 0.2995, andz is 0.0005 to 0.001; and

Na(Y_(x) Yb_(y) Tm_(z))F₄ : x is 0.7 to 0.9, y is 0.0995 to 0.2995, andz is 0.0005 to 0.001.

(Y_(x) Yb_(y) Er_(z))O₂ S: x is 0.7 to 0.9, y is 0.05 to 0.12; z is 0.05to 0.12.

(Y₀.86 Yb₀.08 Er₀.06)₂ O₃ is a relatively efficient up-convertingphosphor material.

For exemplification, but not to limit the invention,ytterbium(Yb)-erbium(Er)-doped yttrium oxysulfides luminesce in thegreen after excitation at 950 nm. These are non-linear phosphors, inthat the ytterbium acts as an "antenna" (absorber) for two 950 nmphotons and transfers its energy to erbium which acts as an emitter(activator). The critical grain size of the phosphor is given by thequantum yield for green emission and the doping level of both Yb and Er,which is generally in the range of about 1 to 10 percent, more usuallyin the range of about 2 to 5 percent. A typical Yb:Er phosphor crystalcomprises about 10-30% Yb and about 1-2% Er. Thus, a phosphor graincontaining several thousand formula units ensures the emission of atleast one or more photons during a typical laser irradiation time.However, the nonlinear relationship between absorption and emissionindicates that intense illumination at the excitation wavelength(s) maybe necessary to obtain satisfactory signal in embodiments employing verysmall phosphor particles (i.e., less than about 0.3 mu m). Additionally,it is usually desirable to increase the doping levels ofactivatoremitter couples for producing very small phosphor particles soas to maximize quantum conversion efficiency.

Inorganic microcrystalline phosphors with rare earth activatorsgenerally have narrow absorption and line emission spectra. The lineemission spectra are due to f--f transitions within the rare earth ion.These are shielded internal transitions which result in narrow lineemission.

In certain applications, such as where highly sensitive detection isrequired, intense illumination can be provided by commercially availablesources, such as infrared laser sources (e.g., continuous wave (CW) orpulsed semiconductor laser diodes). For example, in applications wherethe microcrystalline phosphor particle must be very small and thequantum conversion efficiency is low, intense laser illumination canincrease signal and decrease detection times. Alternatively, someapplications of the invention may require phosphor compositions thathave inherently low quantum conversion efficiencies (e.g., low dopinglevels of activator couple), but which have other desirablecharacteristics (e.g., manufacturing efficiency, ease of derivatization,etc.); such low efficiency up-converting phosphors are preferablyexcited with laser illumination at a frequency at or near (i.e., withinabout 25 to 75 nm) an absorption maximum of the material. The fact thatno other light is generated in the system other than from theup-converting phosphor allows for extremely sensitive signal detection,particularly when intense laser illumination is used as the source ofexcitation radiation. Thus, the unique property of up-conversion ofphoton energy by up-converting phosphors makes possible the detection ofvery small particles of microcrystalline inorganic phosphors. Forpractical implementation of phosphors as ultrasensitive reporters,particularly as intracellular reporters, it is essential that the grainsize of the phosphor be as small as practicable (typically less thanabout 0.3 to 0.1 mu m), for which laser-excited up-converting phosphorsare well-suited.

For example, various phosphor material compositions capable ofup-conversion are suitable for use in the invention are shown in TableI.

                  TABLE I                                                         ______________________________________                                        Phosphor Material Compositions                                                Host Material Absorber Ion                                                                             Emitter Ion                                                                              Color                                     ______________________________________                                        Oxysulfides (O.sub.2 S)                                                       Y.sub.2 O.sub.2 S                                                                           Ytterbium  Erbium     Green                                     Gd.sub.2 O.sub.2 S                                                                          Ytterbium  Erbium     Red                                       La.sub.2 O.sub.2 S                                                                          Ytterbium  Holmium    Green                                     Oxyhalides (OX.sub.y)                                                         YOF           Ytterbium  Thulium    Blue                                      Y.sub.3 OCl.sub.7                                                                           Yterbium   Terbium    Green                                     Fluorides (F.sub.x)                                                           YF.sub.3      Ytterbium  Erbium     Red                                       GdF.sub.3     Ytterbium  Erbium     Green                                     LaF.sub.3     Ytterbium  Holmium    Green                                     NaYF.sub.3    Ytterbium  Thulium    Blue                                      BaYF.sub.5    Ytterbium  Thulium    Blue                                      BaY.sub.2 F.sub.8                                                                           Ytterbium  Terbium    Green                                     Gallates (Ga.sub.x O.sub.y)                                                   YGaO.sub.3    Ytterbium  Erbium     Red                                       Y.sub.3 Ga.sub.5 O.sub.12                                                                   Ytterbium  Erbium     Green                                     Silicates (Si.sub.x O.sub.y)                                                  YSi.sub.2 O.sub.5                                                                           Ytterbium  Holmium    Green                                     YSi.sub.3 O.sub.7                                                                           Ytterbium  Thulium    Blue                                      ______________________________________                                    

In addition to the materials shown in Table I and variations thereof,aluminates, phosphates, and vanadates can be suitable phosphor hostmaterials. In general, when silicates are used as a host material, theconversion efficiency is relatively low. In certain uses, hybridup-converting phosphor crystals may be made (e.g., combining one or morehost material and/or one or more absorber ion and/or one or more emitterion).

Exemplary up-converting phosphors excited at about 980 nm include, butare not limited to: Y₀.80 Yb₀.18 Er₀.02)F₃ ; Y₀.87 Yb₀.13 Tm₀.001)F₃ ;Y₀.80 Yb₀.198 Ho₀.002)F₃ ; Gd₀.80 Yb₀.18 Er₀.02)F₃ ; Gd₀.87 Yb₀.13Tm₀.001)F₃ ; Gd₀.80 Yb₀.198 Ho₀.002)F₃ ; Y₀.86 Yb₀.08 Er₀.06)₂ O₂S;Y₀.87 Yb₀.13 Tm₀.001)₂ O₂ S; Y₀.80 Yb₀.198 Ho₀.002)₂ O₂ S; Gd₀.86Yb₀.08 Er₀.06)₂ O₂ S; Gd₀.87 Yb₀.13 Tm₀.001)₂ O₂ S;Gd₀.80 Yb₀.198Ho₀.002)₂ O₂ S.

Exemplary up-converting phosphors excited at about 1500 nm include, butare not limited to: Y₀.96 Er₀.06)₂ O₂ S; Gd₀.96 Er₀.06)₂ O₂ S.

Preparation of Inorganic Phosphor Labels

Techniques and methods for manufacture of inorganic phosphors has beendescribed in the art. Up-converting phosphor crystals can bemanufactured by those of ordinary skill in the art by various publishedmethods, including but not limited to the following: Yocom et al.,(1971) Metallurgical Transactions 2: 763; Kano et al., (1972). J.Electrochem. Soc., p. 1561; Wittke et al. (1972) J. Appl. Physics 43:595; Van Uitert et al. (1969) Mat. Res. Bull. 4: 381; which areincorporated herein by reference. Other references which may be referredto are:

Jouart J. P. and Mary G. (1990) J. Luminescence 46: 399; McPherson G. L.and Meyerson S. L. (1991) Chem. Phys. Lett. (April) p. 325; Oomen et al.(1990) J. Luminescence 46: 353; NI H and R and SC (1991) Optics Lett. 16(September); McFarlane R. A. (1991)Optics Lett. 16 (September); Koch etal. (1990) Appl. Phys. Lett. 56: 1083; Silversmith et al. (1987) Appl.Phys. Lett. 51: 1977; Lenth W. and McFarlane R. M. (1990) J.Luminescence 45: 346; Hirao et al. (1991) J. Non-crystalline Solids 135:90; McFarlane et al. (1988) Appl. Phys. Lett. 52: 1300, incorporatedherein by reference).

In general, inorganic phosphor particles are milled to a desired averageparticle size and distribution by conventional milling methods known inthe art, including milling in a conventional barrel mill with zirconiaand/or alumina balls for periods of up to about 48 hours or longer.Phosphor particles used in binding assays are typically about 3.0 to0.01 μm in diameter (or along the long axis if non-spherical), moreusually about 2.0 to 0.1 μm in size, and more conveniently about 1.0 to0.3 μm in size, although phosphor particles larger or smaller than thesedimensions may be preferred for certain embodiments. Phosphor particlesize is selected by the practitioner on the basis of the desiredcharacteristics and in accordance with the guidelines provided herein.Fractions having a particular particle size range may be prepared bysedimentation, generally over an extended period (i.e., a day or more)with removal or the desired size range fraction after the appropriatesedimentation time. The sedimentation process may be monitored, such aswith a Horiba Particle Analyzer.

However, milling crystalline materials has several weaknesses. Withmilling, the particle morphology is not uniform, as milled particlesresult from random fracture of larger crystalline particles. Since thesensitivity of a detection assay using up-converting inorganic phosphorsdepends on the ability to distinguish between bound and unbound phosphorparticles, it is preferable that the particles be of identical size andmorphology. Size, weight, and morphology of up-convertingmicrocrystalline phosphor particles can affect the number of potentialbinding sites per particle and thus the potential strength of particlebinding to reporter and/or analyte. Monodisperse submicron sphericalparticles of uniform size can be generated by homogeneous precipitationreactions at high dilutions. For example, small yttrium hydroxycarbonate particles are formed by the hydrolysis of urea in a diluteyttrium solution. Similarly, up-converting inorganic phosphors can beprepared by homogeneous precipitation reactions in dilute conditions.For example, (Y₀.86 Yb₀.08 Er₀.06)₂ O₃ was prepared as monodispersespherical particles in the submicron size range by precipitation.

However, after precipitation it is typically necessary to anneal theoxide in air at about 1500° C., which can cause faceting of thespherical particles which can generate aggregate formation. Faceting canbe substantially reduced by converting the small spherical particles ofthe oxide or hydroxy carbonate precursor to the oxysulfide phase byincluding a polysulfide flux for annealing. Using this technique, highlyefficient oxysulfide particles in the 0.3 to 0.4 μm diameter range wereprepared as a dispersion in water. Frequently, sonication can be used toproduce a monodisperse mixture of discrete spherical particles. Afterfractionation and coating, these particles can be used as up-convertingreporters. Furthermore, this general preparative procedure is suitablefor preparing much smaller phosphor particles (e.g., 0.1 μm diameter orsmaller), which may be advantageous for various assay formats.

Frequently, such as with phosphors having an oxysulfide host material,the phosphor particles are preferably dispersed in a polar solvent, suchas acetone or DMSO and the like, to generate a substantiallymonodisperse emulsion (e.g., for a stock solution). Aliquots of themonodisperse stock solution may be further diluted into an aqueoussolution (e.g., a solution of avidin in buffered water or bufferedsaline).

It was found that washing phosphors in acetone or DMSO improvedsuspendability of inorganic phosphor particles in water. In particular,the phosphor particles prepared with polysulfide flux are preferablyresuspended and washed in hot DMSO and heated for about an hour in asteam bath then allowed to cool to room temperature under continuousagitation. The phosphor particles may be pre-washed with acetone(typically heated to boiling) prior to placing the particles in theDMSO. Hot DMSO-treated phosphors were found to be reasonably hydrophilicand form stable suspensions. A Microfluidizer™ (Microfluidics Corp.) canbe used to further improve the dispersion of particles in the mixture.DMSO-phosphor suspensions can be easily mixed with water, preferablywith small amounts of surfactant present. In general, polysaccharides(e.g., guar gum, xanthan gum, gum arabic, alginate, guaiac gum) can beused to promote deaggregation of particles. In a variation, particlesare washed in hot DMSO and serially diluted into a 0.1% aqueous gumarabic solution, which appears to virtually eliminate water dispersionproblems of phosphors.

Resuspended phosphors in organic solvent, such as DMSO, are typicallyallowed to settle for a suitable period (e.g., about 1-3 days), and thesupernatant which is typically turbid is used for subsequentconjugation.

Ludox™ is a colloidal silica dispersion in water with a small amount oforganic material (e.g., formaldehyde, glycols) and a small amount ofalkali metal. Ludox™ and its equivalents can be used to coatup-converting phosphor particles which can subsequently be fired to forma ceramic silica coating which cannot be removed from the phosphorparticles, but which can be readily silanized with organofunctionalsilanes (containing thiol, primary amine, and carboxylic acidfunctionalities) using standard silanization chemistries (Arkles, B.,in: Silicon Compounds: Register and Review; 5th Edition (1991);Anderson, R. G., Larson, G. L., and Smith, C., eds.; p. 59-64, HulsAmerica, Piscataway, N.J.).

Phosphor particles can be coated or treated with surface-active agents(e.g., anionic surfactants such as Aerosol OT) during the millingprocess or after milling is completed. For example, particles may becoated with a polycarboxylic acid (e.g., Additon XW 330, Hoechst, Frart,Germany or Tamol, see Beverloo et al. (1992) op.cit.) during milling toproduce a stable aqueous suspension of phosphor particles, typically atabout pH 6-8. The pH of an aqueous solution of phosphor particles can beadjusted by addition of a suitable buffer and titration with acid orbase to the desired pH range. Depending upon the chemical nature of thecoating, some minor loss in conversion efficiency of the phosphor mayoccur as a result of coating, however the power available in a laserexcitation source can compensate for such reduction in conversionefficiency and ensure adequate phosphor emission.

In general, preparation of inorganic phosphor particles and linkage tobinding reagents is performed essentially as described in Beverloo etal. (1992) op.cit., and Tanke U.S. Pat. No. 5,043,265 incorporated byreference in their entirety herein. Alternatively, a water-insolublepolyfunctional polymer which exhibits glass and melt transitiontemperatures well above room temperature can be used to coat theup-converting phosphors in a nonaqueous medium. For example, suchpolymer functionalities include: carboxylic acids (e.g., 5% acrylicacid/95% methyl acrylate copolymer), amine (e.g., 5% aminoethylacrylate/95% methyl acrylate copolymer) reducible sulfonates (e.g., 5%sulfonated polystyrene), and aldehydes (e.g., polysaccharidecopolymers). The phosphor particles are coated with water-insolublepolyfunctional polymers by coacervative encapsulation in nonaqueousmedia, washed, and transferred to a suitable aqueous buffer solution toconduct the heterobifunctional crosslinking to a protein (e.g.,antibody) or polynucleotide probe molecule. An advantage of usingwater-insoluble polymers is that the polymer microcapsule will notmigrate from the surface of the phosphor upon aging the encapsulatedphosphors in an aqueous solution (i.e., improved reagent stability).Another advantage in using copolymers in which the encapsulating polymeris only partially functionalized is that one can control the degree offunctionalization, and thus the number of biological probe moleculeswhich can be attached to a phosphor particle, on average. Since thesolubility and coacervative encapsulation process will depend on thedominant nonfunctionalized component of the copolymer, thefunctionalized copolymer ratio can be varied over a wide range togenerate a range of potential crosslinking sites per phosphor, withouthaving to substantially change the encapsulation process.

A preferred functionalization method employs heterobifunctionalcrosslinkers that can be made to link the biological macromolecule probeto the insoluble phosphor particle in three steps: (1) bind thecrosslinker to the polymer coating on the phosphor, (2) separate theunbound crosslinker from the coated phosphors, and (3) bind thebiological macromolecule to the washed, linked polymer-coated phosphor.This method prevents undesirable crosslinking interactions betweenbiological macromolecules and so reduces irreversible aggregation asdescribed by Tanke et al. Examples of suitable heterobifunctionalcrosslinkers, polymer coating functionalities, and linkable biologicalmacromolecules include, but are not limited to:

    ______________________________________                                        Coating   Heterobifunctional                                                                              Biological                                        Functionality                                                                           Crosslinker       Macromolecule                                     ______________________________________                                        carboxylate                                                                             N-hydroxysuccimide                                                                              Proteins (e.g.,                                             1-ethyl-3-(3-dimethylamino                                                                      Ab, avidin)                                                 propyl)-carbodiimide (EDC)                                          primary amine                                                                           N-5-azido-2-nitrobenzoyl                                                                        All having 1.sup.0  amine                                   oxysuccimide (ANB-NOS)                                                        N-succinimidyl (4-iodoacetyl)                                                 aminobenzoate (SIAB)                                                thiol (reduced                                                                          N-succinimidyl (4-iodoacetyl)                                                                   Proteins                                          sulfonate)                                                                              aminobenzoate (SIAB)                                                ______________________________________                                    

Binding Assays

Up-converting phosphors and up-converting organic dyes are used asreporters (i.e., detectable markers) to label binding reagents, eitherdirectly or indirectly, for use in binding assays to detect andquantitate the presence of analyte(s) in a sample. Binding reagents arelabeled directly by attachment to up-converting reporters (e.g., surfaceadsorption, covalent linkage). Binding reagents which can be directlylabeled include, but are not limited to: primary antibodies (i.e., whichbind to a target analyte), secondary antibodies (i.e., which bind to aprimary antibody or prosthetic group, such as biotin or digoxygenin),Staphlococcus aureus Protein A, polynucleotides, streptavidin, andreceptor ligands. Binding reagents can also be indirectly labeled; thus,a primary antibody (e.g., a rabbit anti-erb-B antibody) can beindirectly labeled by noncovalent binding to a directly labeled secondantibody (e.g., a goat anti-rabbit antibody linked to an up-convertinginorganic phosphor). Quantitative detection of the analyte-probe complexmay be conducted in conjunction with proper calibration of the assay foreach probe employed. A probe is conveniently detected under saturatingexcitation conditions using, for example, a laser source or focusedphotodiode source for excitation illumination.

Specific binding assays are commonly divided into homogeneous andheterogeneous assays. In a homogeneous assay, the signal emitted by thebound labeled probe is different from the signal emitted by the unboundlabeled probe, hence the two can be distinguished without the need for aphysical separation step. In heterogeneous assays, the signal emittedfrom the bound and unbound labeled probes is identical, hence the twomust be physically separated in order to distinguish between them. Theclassical heterogeneous specific binding assay is the radioimmunoassay(RIA) (Yalow et al. (1978) Science 200: 1245, which is incorporatedherein by reference). Other heterogeneous binding assays include theradioreceptor assay (Cuatrecasas et al. (1974) Ann. Rev. Biochem. 43:109), the sandwich radioimmunoassay (U.S. Pat. No. 4,376,110, which isincorporated herein by reference), and the antibodylectin sandwich assay(EP 0 166 623, which is incorporated herein by reference). Heterogeneousassays are usually preferred, and are generally more sensitive andreliable than homogeneous assays.

Whether a tissue extract is made or a biological fluid sample is used,it is often desirable to dilute the sample in one or more diluents thatdo not substantially interfere with subsequent assay procedures.Generally, suitable diluents are aqueous solutions containing a buffersystem (e.g., 50 mM NaH₂ PO₄ or 5-100 mM Tris, pH 4-pH 10),non-interfering ionic species (5-500 mM KCl or NaCl, or sucrose), andoptionally a nonionic detergent such as Tween. When the sample to beanalyzed is affixed to a solid support, it is usually desirable to washthe sample and the solid support with diluent prior to contacting withprobe. The sample, either straight or diluted, is then analyzed for thediagnostic analyte.

In the general method of the invention, an analyte in a sample isdetected and quantified by contacting the sample with a probe-labelconjugate that specifically or preferentially binds to an analyte toform a bound complex, and then detecting the formation of bound complex,typically by measuring the presence of label present in the boundcomplexes. A probe-label conjugate can include a directly labeledanalyte-binding reagent (e.g., a primary antibody linked to anup-converting phosphor) and/or an indirectly labeled analyte-bindingreagent (e.g., a primary antibody that is detected by a labeled secondantibody, or a biotinylated polynucleotide that is detected by labeledstreptavidin). The bound complex(es) are typically isolated from unboundprobe-label conjugate(s) prior to detection of label, usually byincorporating at least one washing step, so as to remove backgroundsignal attributable to label present in unbound probe-labelconjugate(s). Hence, it is usually desirable to incubate probe-labelconjugate(s) with the analyte sample under binding conditions for asuitable binding period.

Binding conditions vary, depending upon the nature of the probe-labelconjugate, target analite, and specific assay method. Thus, bindingconditions will usually differ if the probe is a polynucleotide used inan in situ hybridization, in a Northern or Southern blot, or in solutionhybridization assay. Binding conditions will also be different if theprobe is an antibody used in an in situ histochemical staining method ora Western blot (Towbin et al. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76:4350, incorporated herein by reference). In general, binding conditionsare selected in accordance with the general binding methods known in theart. For example, but not for limitation, the following bindingconditions are provided for general guidance:

For antibody probes:

10-200 mM Tris, pH 6-8; usually 100 mM Tris pH 7.5

15-250 mM NaCl; usually 150 mM NaCl

0.01-0.5 percent, by volume, Tween 20

1 percent bovine serum albumin

4°-37° C.; usually 4° to 15° C.

For polynucleotide probes:

3-10×SSC, pH 6-8; usually 5×SSC, pH 7.5

0-50 percent deionized formamide

1-10×Denhardt's solution

0-1 percent sodium dodecyl sulfate

10-200 μg/ml sheared denatured sahnon sperm DNA

20°-65° C., usually 37°-45° C. for polynucleotide probes longer than 50bp, usually 55°-65° C. for shorter oligonucleotide probes

Additional examples of binding conditions for antibodies andpolynucleotides are provided in several sources, including: Maniatis etal., Molecular Cloning: A Laboratory Mantual (1989), 2nd Ed., ColdSpring Harbor, N.Y. and Berger and Kimmel, Methods in Enzoymlogy, Volume152 Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,San Diego, Calif.; Young and Davis (1983) Proc. Natl. Acad. Sci.(U.S.A.) 80: 1194, which are incorporated herein by reference. When theprobe is a receptor ligand, such as IL-2, β-interferon, or otherpolypeptide hormones, cytokines, or lymphokines, suitable bindingconditions generally are those described in the art for performing therespective receptor-ligand binding assay.

Various examples of suitable binding conditions useful in immunoassaysand immunohistochemistry are discussed, for example, in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988), whichis incorporated herein by reference. In general, suitable bindingconditions for immunological reactions include an aqueous binding buffercontaining a salt (e.g., 5-500 mM NaCl or KCl), a buffer (e.g., Tris orphosphate buffer at pH 4-10), and optionally a nonionic detergent (e.g.,Tween). In some embodiments, proteinase inhibitors or stabilizers may beincluded. The binding reactions are conducted for a suitable bindingperiod, which, for antibody reactions, are typically at least about 1 to5 minutes, preferably at least about 30 minutes to several hours,although typically less than about 24 hours, more preferably less thanabout a few hours or less. Binding reactions (including washes) aretypically carried out a temperature range of about 0° C. to about 45°C., preferably about 4° C. to about 20°-25° C.

Binding assays, which include in situ hybridization, in situ bindingassays, and immunohistochemical staining, are usually performed by firstincubating the sample with a blocking or prehybridization solution,followed by incubating the sample with probe under binding conditionsfor a suitable binding period, followed by washing or otherwise removingunbound probe, and finally by detecting the presence, quantity, and/orlocation of bound probe. The step of detecting bound probe can beaccomplished by detecting label, if the probe is directly labeled, or byincubating the bound complex(es) with a second binding reagent (e.g.,streptavidin) that is labeled and which binds to the probe, thusaccomplishing indirect labeling of the probe.

Up-converting labels are attached to probe(s) or second binding reagentsthat specifically or preferentially bind to probe(s) by any of thevarious methodologies discussed herein. Additionally, up-convertingphosphor particles can be encapsulated in microspheres and coated with aprobe (e.g., a specific antigen or antibody) for use as a labeled probein an immunodiagnostic assay or nucleic acid hybridization assay todetect an analyte in a sample, such as the presence of an antibody,virus, or antigen in a bloom serum sample, according to the method ofHari et al. (1990) Biotechniques 9:342, which is incorporated herein byreference. Microencapsulation of phosphor can be accomplished in severalways known in the art, including coating the phosphor with a monomersolution and polymerizing the monomer to generate a polymer shellencasing the phosphor particle. Phosphor particles embedded in a polymercoating, such as a gel coating, can be functionalized (e.g., with aminogroups) for covalent attachment to a binding component.

Similarly, up-converting phosphor particles can be coated with probedirectly, either by surface adsorption, by multiple hydrogen bonding, byelectrostatic interaction, by van der Waals binding, or by covalentlinkage to a functional group on a functionalized inorganic phosphorparticle (e.g., a vitroceramic phosphor), for example, by linking anamino acid side-chain amine or carboxylate group of a probe protein to acarboxylate or amine group, respectively, on a functionalized phosphorparticle.

In certain embodiments, such as where stearic and/or charge interferenceof a bulky up-converting phosphor particle inhibits binding of thelinked binding reagent to a target, it is desirable to incorporate amolecular spacer between the phosphor particle and the binding reagent.For example, a derivatized microencapsulated phosphor or vitroceramicphosphor may be conjugated to a heterobifunctional reagent having a--(CH₂)_(n) -- spacer, where n is usually an integer from about 2 toabout 50, between terminal functional groups. Similarly, phosphors maybe directly derivatized with derivatizing agents (e.g.,omega-functionalized silanes) having long intramolecular spacer chains,wherein a functional group reactive with a desired binding reagent isseparated from the surface of the phosphor by a spacer of usually atleast about 15 Å (i.e., the equivalent of about 10 --CH₂ --straight-chain groups). In some embodiments, labels are attached byspacer arms of various lengths to reduce potential stearic hindrance.Multiple layers of spacer arms may also be used (e.g., multiple layersof streptavidin-biotin linkages).

Multiple Analyte Detection

Since up-converting phosphors can be differentiated on the basis of theexcitation and/or emission wavelength spectra, up-converting phosphorscan be used to detect and discriminate multiple analyte targets, suchas, for example, cell surface antigens or soluble macromolecules.

For example, streptavidin, avidin, or another linker macromolecule(e.g., antidigoxigenin antibody) are attached, respectively, to each oftwo different phosphors (for illustration, designated here as Phosphor#1 and Phosphor #2) which differ in their absorption and/or emissionspectra so as to facilitate discrimination of the two phosphors based onabsorption and/or emission wavelengths; e.g., one phosphor may emit inthe blue and the other may emit in the green. For example and notlimitation, Na(Y₀.80 Yb₀.18 Er₀.02)F₄ emits predominantly in the green,and Na(Y₀.73 Yb₀.27 TM₀.001)F₄ emits predominantly in the blue, and thusthese two phosphors may be discriminated on the basis of theirphosphorescent emissions. Alternatively, two phosphors may produceessentially similar emission spectra but may have different excitationwavelengths which provide a basis for their discrimination in multipleanalyte detection. A first binding component (e.g., an antibody) thatbinds specifically to a first analyte species (e.g., a lymphocyte CD4antigen) and incorporates biotinyl moieties which may be bound bystreptavidin-Phosphor #1 conjugates can be used to quantitatively detectthe presence of a first analyte in a sample (e.g., a serum sample) bymeasuring phosphorescence of Phosphor #1 in analyte-binding componentcomplexes. A second binding component (e.g., a probe polynucleotide)that binds specifically to a second analyte species (e.g., an HIV-1sequence) and incorporates digoxygenin moieties (e.g.,11-UTP-digoxygenin) which may be bound by antiligoxigenin-Phosphor #2conjugates can be used to quantitatively detect the presence of a secondanalyte in the sample by measuring phosphorescence of Phosphor #2 inanalyte-binding component complexes. Thus, by simultaneously orcontemporaneously detecting the presence of multiple phosphor reportershaving differentiable signal characteristics, multiple analytes may bequantitatively detected in a single sample.

Sandwich Binding Assays

Up-converting phosphors labels can be used as reporters for sandwichbinding assays (U.S. Pat. No. 4,376,110, which is incorporated herein byreference). For example, a magnetic bead, such as a superparamagneticimmunobead or functionalized magnetizable polymer particle(Polysciences, Inc., Warrington, Pa.), can serve as the solid substratewhich has an immobilized first binding component (e.g., an antibody, apolynucleotide, or a lectin) that binds to a first epitope (i.e., abinding locus: an antigenic determinant, sugar moiety, chemicalsubstituent, or nucleotide sequence) of an analyte. The analyte binds tothe first binding component and also to a second binding component(e.g., an antibody, a lectin, or a polynucleotide) which binds to asecond epitope of the analyte. Thus, the analyte bridges the two bindingcomponents to form a sandwich complex which is immobilized with respectto the solid substrate. The second binding component typically has anattached or incorporated label, such as a biotinyl group which can bebound to a streptavidin-coated up-converting phosphor. Alternatively,the second binding component can be linked directly to an up-convertingphosphor, such as through a covalent linkage with a functionalizedvitroceramic up-converting phosphor.

The sandwich complex comprises the first binding component, an analyte,and the second binding component, which is labeled, either directly orindirectly, with an up-converting reporter. The sandwich complex is thusimmobilized on the solid substrate, although the solid substrate itselfmay be mobile (e.g., a superparamagnetic bead circulating in a sampleslurry). The presence and amount of analyte(s) can be quantitativelymeasured by detecting the presence of up-converting reporter in sandwichcomplexes.

For example, a solid substrate may have a plurality of distinct speciesof first binding component (e.g., an array of different oligopeptidesaffixed to a solid support). One or more of the species of first bindingcomponent may bind to a particular analyte (e.g., a muscarinic receptor)in an analyte solution that is in contact with the solid support.Binding of the analyte to one or more of the first binding componentspecies may then be detected with a second binding component (e.g., ananti-muscarinic receptor antibody) labeled with an up-convertingphosphor (either directly or through a biotinylated secondary antibody).

Solid substrates can be attached to a first binding component which canbind more than one distinct analyte (e.g., may be immunocrossreactive orpolyspecific) and/or can be attached to multiple first binding componentspecies which can bind multiple distinct analytes. Similarly, multiplesecond binding component species with binding specificities forparticular analytes can be employed. When multiple second bindingcomponent species are employed, it is typically desirable to label eachsecond binding component species with a unique up-converting label thatcan be distinguished on the basis of its absorption and/or emissionproperties.

It is possible to use different absorbers in combination with variousemitters to produce a collection of phosphors having severaldifferentiable combinations of excitation and emission spectra. Forexample but not limitation, six differentiable phosphors may begenerated from two absorbers and three emitters. A first absorber, A₁,has an excitation wavelength of λ_(A1), a second absorber, A₂ has anexcitation wavelength of λ_(A2), a first emitter, E₁, has an emissionline at λ_(E1), a second emitter, E₂ has an emission line at λ_(E2), anda third emitter, E₃, has an emission line at λ_(E3). The six phosphorsmay be differentiated and the signal from each individually quantitatedby illuminating the sample with an excitation wavelengthλ_(A1) anddetecting separately the emitted radiation at λ_(E1), λ_(E2), andA_(E3), and separately illuminating the sample with λ_(A2) and detectingseparately the emitted radiation at λ_(E1), λ_(E2), and λ_(E3). Table IIshows the various absorber:emitter combinations and their excitation andemission wavelengths.

                  TABLE II                                                        ______________________________________                                        Absorber:Emitter Combination                                                                     Excitation                                                                              Emission                                         ______________________________________                                        A1:E1              A1        E1                                               A1:E2              A1        E2                                               A1:E3              A1        E3                                               A2:E1              A2        E1                                               A2:E2              A2        E2                                               A2:E3              A2        E3                                               ______________________________________                                    

Of course, additional absorber:emitter combinations are possible toprovide more than six differentiable phosphor labels.

It is also possible to utilize solid substrates of different types whichmay be distinguished (e.g., by size, color, density, magneticproperties, shape, charge) so that a particular type of solid substrateis associated with a particular species of first binding component.

For example and not limitation, the following three brief examples areprovided to explicate further possible applications of multiple analytesandwich assay methods.

Substrate Differentiation

The following example describes the use of distinguishable substratetypes to detect the presence of specific immunoglobulin idiotypes in asample (e.g., a blood serum sample taken from a patient) which canprovide diagnostic information about the immune status of a patient(e.g., is a patient seroreactive with a particular antigen).

Large superparamagnetic beads are conjugated to an immunogenicHerpesvirus Type II envelope glycoprotein, medium-sizedsuperparamagnetic beads are conjugated to HIV gp120 glycoprotein, andsmall superparamagnetic beads are conjugated to an immunogeniccytomegalovirus envelope glycoprotein. A serum sample is taken from apatient and is incubated with a mixture of the superparamagnetic beadsunder binding conditions to permit specific binding of immunoglobulinsin the sample with the three immobilized viral glycoprotein species. Thesuperparamagnetic beads are separated from the sample to removenon-specifically bound immunoglobulin and incubated with up-convertingphosphor particles coated with Staphylococcus aureus Protein A, whichbinds to IgG, under binding conditions. Supeiparamagnetic beads havingspecifically bound IgG are thus labeled with the phosphor-Protein Aconjugate. Large, medium, and small superparamagnetic beads are thenseparately illuminated with phosphor excitation electromagneticradiation and time-gated emitted phosphorescence is detected. Backgroundattributable to non-specific binding, if any, is determined andsubtracted using internal standard beads (bovine serum albumin coatedsuperparamagnetic beads) and positive and negative control serumsamples. The intensity of phosphorescence associated with the large,medium, and small beads provides a measure of the amount of antibodiesin the sample which are reactive with the Herpesvirus Type II envelopeglycoprotein, HIV gp120 glycoprotein, and cytomegalovirus envelopeglycoprotein, respectively. This information can be used to determinewhether an individual patient has been infected with the HIV-1, humanCMV, and/or Herpes Simplex Type II viruses.

Phosphor Differentiation

The following example describes the use of differentiable up-convertingphosphors to detect the presence and relative abundance of particularisoforms of human APP (amyloid precursor protein) in a serum or brainbiopsy sample. Various isoforms of APP arise in the brain as aconsequence of alternative exon usage and/or alternative proteolyticprocessing pathways. Thus, although all APP isoforms may share a common,hypothetical epitope (X), a particular APP isoform may have a uniqueepitope (Y), while another APP isoform has a unique epitope (Z). It ispossible that the relative abundance of a particular APP isoform in asample may be of predictive value or may be pathognomonic forAlzheimer's Disease.

Superparamagnetic beads are conjugated to an antibody that bindsspecifically to a common APP epitope (X) shared by all isoforms. Aspecific antibody reactive with the unique Y epitope is labeled withPhosphor #1, which is excited by wavelength λ₁ and emits in a wavelengthspectrum centered in the blue. A specific antibody reactive with theunique Z epitope is labeled with Phosphor #2, which is excited by awavelength λ₂ and emits in a wavelength spectrum centered in the green.A sample containing APP isoforms is incubated with the superparamagneticbeads and labeled specific antibodies under binding conditions. Thesuperparamagnetic beads are retrieved from the sample, eitherindividually or in bulk. The beads are illuminated with wavelength λ₁and blue light emission is detected and measured, and illuminated withλ₂ and green light emission is detected and measured. The intensity ofλ₁ -induced blue emission is a measure of the APP isoform(s) having theY epitope, while the intensity of the λ₂ -induced green emission is ameasure of the APP isoform(s) having the Z epitope. If the emissionsfrom two phosphors are readily distinguishable, λ₁ and λ₂ may beidentical. The standardized relative intensities of the two phosphorsprovides a measure of the relative abundance of the APP isoform(s)containing the Y or Z epitopes.

Phosphor and Substrate Differentiation

The following example describes the use of differentiable up-convertingphosphors in conjunction with distinguishable substrate types to detectthe presence and relative abundance of particular T lymphocytesubpopulations in a blood sample taken from an individual. Althoughdescribed here with reference to detecting T cell subpopulations,analyte multiplexing (i.e., detecting and/or characterizing multipleanalytes in a sample by using various solid substrate types and/orup-converting phosphor labels) is believed to be a generally applicablemethod.

Large superparamagnetic beads are conjugated to an anti-CD4 antibody,medium-sized superparamagnetic beads are conjugated to anti-CD8antibody, and small superparamagnetic beads are conjugated to ananti-CD28 antibody. An antibody that specifically binds to the CD2antigen is labeled with an up-converting phosphor that has an excitationwavelength λ₁ and emits in the red. An antibody that specifically bindsto the CD45R antigen is labeled with an up-converting phosphor that hasan excitation wavelength λ₂ and emits in the green. An antibody thatspecifically binds to the CDw60 antigen is labeled with an up-convertingphosphor that has an excitation wavelength s and emits in the blue.

A blood (or serum, sputum, urine, feces, biopsy tissue, etc.) sample istaken from a patient and is incubated with a mixture of thesuperparamagnetic beads and phosphor-labeled antibodies under bindingconditions to permit specific binding of cells in the blood sample withthe three bead-immobilized antibody species and the threephosphor-labeled antibody species. After antigen-antibody bindingoccurs, the superparamagnetic beads are segregated and examined, eithersequentially or simultaneously, by illumination with λ₁, λ₂, and λ₃, andquantitative detection of red, green, and blue emissions, respectively.For example, the intensity ofλ₁ -induced red light emission associatedwith the large beads is a rough measure of the amount of cells havingboth CD4 and CD2 surface antigens and/or the relative abundance of thosesurface antigens (e.g., there may be very few CD4+ cells that have CD2,but those few cells may have a large amount of CD2 antigen, and hence alarge CD2 phosphorescent signal). Similarly, the intensity of λ₂-induced green light associated with the large beads is a rough measureof the amount of cells having both CD4 and CD45R surface antigens and/orthe relative abundance of those surface antigens in a sample.

In this manner, an analyte sample, such as a blood sample, can be"fingerprinted" for the presence and relative distribution(s) (e.g.,cosegregation and/or correlation) of various analyte species. Such ananalyte fingerprint may be used for providing diagnostic or therapeuticinformation, for example, as to measuring a patient's immune status ormeasuring response to chemotherapy directed against a particular bloodcell subset. Similar analyte fingerprints can be used to type pathogenicorganisms and viruses, as well as to order polynucleotide sequences forgene mapping and/or sequencing. Superparamagnetic beads which can bedifferentiated based on size, shape, color, or density can bemagnetically trapped individually and scanned with appropriateexcitation illumination(s) and phosphor emission(s) characteristic ofparticular analytes detected. For example, a unitary detector cansimultaneously or contemporaneously trap the superparamagnetic bead froma suspension, determine the bead type (size, shape, and/or color), andscan for presence and abundance of particular phosphors (by illuminatingwith excitation wavelength(s) and detecting emitted wavelengths). Byperforming binding assays under dilute conditions wherein an average ofone analyte or less (e.g., lymphocyte) is bound per microbead, it ispossible to type cells individually (e.g., determine the abundance ofCD45R on each individual CD4⁺ cell) and thus generate more preciselymphocyte subpopulation definitions. Biotinylated magnetic beads canalso be used to monitor the kinetics of binding streptavidin to phosphorparticles and/or to segregate or purify streptavidin-coatedup-converting phosphor particles from a reaction. Thus, streptavidin andup-converting phosphor particles are mixed in a reaction vessel underbinding conditions for forming streptavidin-coated phosphor particles.After a suitable binding period, unbound streptavidin may be removed(e.g., by centrifugation wherein phosphor particles are collected as thepellet, unbound streptavidin in the supernatant is decanted, and thepellet is resuspended), biotinylated magnetic beads are added to theremaining phosphor suspension in binding conditions, andstreptavidin-coated phosphor particles are recovered bound to thebiotinylated magnetic beads.

Photophysical Catalysis by Up-Converting Phosphors

Other applications of the invention employ phosphors as a photophysicalcatalyst linked to a probe, where the radiation emitted by the phosphoris used, typically in conjunction with a dye molecule, to producelocalized intense electromagnetic radiation in an area adjacent to theprobe for various purposes other than detection (e.g., cytotoxicity,ionization of chemical species, mutagenesis, etc.). For example, anantibody that specifically binds to a cell surface antigen, such as aCD8 antigen on a CD8⁺ lymphocyte, may be used as a probe linked to aup-converting phosphor to localize the phosphor to CD8⁺ lymphocytes. Asample containing CD8⁺ lymphocytes can be incubated with the anti-CD8⁺probe-phosphor conjugate and irradiated with an excitation wavelength(e.g., from an infrared laser diode), resulting in emission ofup-shifted photons (i.e., higher frequency electromagnetic radiation) inthe vicinity of CD8⁺ lymphocytes to which the anti-CD8⁺ probe-phosphorconjugate has bound. The emitted radiation may be of a wavelength thatis directly mutagenic and/or cytotoxic (e.g., ultraviolet radiation thatcan lead to formation of thymine dimers, 760-765 nm light is alsobelieved to produce chromosomal damage) or may be of a wavelength thatcan cause a photolytic decomposition of a chemical present in theenvironment, leading to local formation of reactive species that maydamage adjacent cells (e.g., photodecomposition of buckminsterfullerene,C₆₀, to C₅₈ and C₂, may produce free radicals that may cause lipidperoxidation of cell membranes).

Since phosphor-emitted radiation is isotropic, it is generally desirableto physically separate targets (e.g., CD8⁺ lymphocytes) from non-targets(e.g., CD8⁻ lymphocytes) prior to excitation irradiation, so thatundesirable damage to non-targets by isotropic emission(s) (i.e.,"secondary damage") is avoided. Physical separation may be accomplishedby various means, including but not limited to: (1) performingexcitation irradiation on a dilute suspension of target and non-targetcells, wherein the mean distance separating individual cells issufficient to reduce secondary damage to non-targets, and (2) employinghydrodynamic focusing to pass cells (both targets and non-targets)single file through an illumination zone (e.g., as in afluorescence-activated cell sorter or the like). Thus, an up-convertingphosphor linked to an anti-DC8⁺ antibody can be used to selectivelydamage CD8⁺ lymphocytes in a lymphocyte sample, where (1) the phosphoremits at a wavelength that is either directly cytotoxic and/or (2) thephosphor emits at a wavelength that produces reactive chemical speciesby photocatalysis of a compound present in the sample (e.g., a samplecan be doped with buckminsterfullerene).

Instead of using the emitted radiation directly for photocatalyticaction on tissue or tumors, an excited form of oxygen, so called singletexcited oxygen (O2'Δg) can be generated by energy transfer from a dyesensitizer to dissolved molecular oxygen. This scheme makes use of thetissue penetrating power of near-infrared radiation (red and ultraredregion light, including 970 nm) which reaches the inorganicup-converting phosphor. Two of the infrared photons are converted eitherinto a red, green, or blue photon depending on the absorption spectrumof the sensitizer dye. The dye is excited by the up-converted radiationinto a triplet state which transfers its energy to a dissolved molecularoxygen molecule to yield an excited (singlet) oxygren molecule. Thecytotoxic activity of singlet oxygen is well documented in photodynamictherapy and other biomedical applications (see, Wagnieres et al. (19-21Jan. 1990) Future Directions and Applications of photodynamic Therapy,pp. 249, SPIE Institutes for Advanced Optical Technologies, Society ofPhoto-Optical Instrumentation Engineers, Box 10, Bellingham, Wash.98277; Pelegrin et al. (1991) Cancer 67: 2529; Wagnieres et al. (24-25May 1991) Future Directions and Applications of Photodynamic Therapy,pp. 219; Folli et al. (Dec. 17, 1991) Fluoresceine Clinique 4;Braichotte et al. (May 1991) ENT-Clinic, Lausanne, Switzerland).

In this application the up-converting phosphor is mixed or laced with asensitizing dye such as methylene blue, rose bengal or phthalocyaninederivatives, such as Zn-phthalocyanine. In the first and third case ared-emitting phosphor is used, whereas for rose bengal a green-emittingphosphor is best suited. The phthalocyanine derivatives are ideallysuited for this purpose because of their total insolubility in aqueousor biological solutions. These dyes therefore stay in close proximity tothe emitters so that the specificity of the cellsurface-reporter/probe/dye complex becomes the limiting factor. In thiscase, specialized combinations of reporter/probe/dye formulationspreferably in the 0.1 to 0.3-micron size range must be synthesized inorder to enable efficient energy transfer: first, up-converted radiationis absorbed by the dye as completely as possible; and second, the dyeexcited energy (triplet state) is transferred to dissolved molecularoxygen. Both processes are very efficient if the absorption spectrum ofthe sensitizer dye is matched to the up-converted radiation. This schemepresents a step beyond the traditional photodynamic therapy methods inthat the red light can be used both for tricking and diagnostic as wellas for therapeutic purposes after up-converting thus necessitating onlyone (infrared) light source at about 1000 nm. A further advantage is thegreater range within biological samples of the infrared radiationcompared to other known photodynamic therapy excitation schemes (750-850nm).

For embodiments employing up-converting phosphors as photophysicalcatalysts, it is generally desirable that: (1) the wavelength(s) of theexcitation radiation do not produce significant photocatalysis of thesubstrate compound, (2) the wavelength(s) of the excication radiationare not directly cytotoxic or mutagenic, and (3) the emitted radiationis directly cytoloxic and/or is of an appropriate wavelength to producea biologically effective amount of photodecomposition of a substratecompound (e.g., buckminsterfullerene, psoralen, compounds containingazide substituents or other photoactivated groups). Alternatively,histidine side chains of polypeptides can be oxidized by light in thepresence of dye sensitizers, such as methylene blue or rose bengal(Proteins Structures and Molecular Principies, (1984) Creighton (ed.),W. H. Freeman and Company, New York; Introduction to Protein Structure,(1991), C. Branden and J. Tooze, Garland Publishing, New York, N.Y.,which are incorporated herein by reference). Thus, for example,up-converting phosphors linked to anti-CD8 antibodies can be used asphotophysical catalysts to produce selective, localized damage to CD8⁺lymphocytes. In accordance with the invention, essentially any antibodycan be linked to an appropriate up-converting phosphor, either directlyor by conjugation to protein A which may then bind the immunoglobulin.Thus, the up-converting photophysical catalysts of the invention may beused to target essentially any desired antigen or cell type that can bedistinguished by the presence of an identified antigen.

Up-converting Chelates

Certain applications require small reporters. For example, thetransport, ability to stay in suspension, the bonding dynamics, and thetendency toward removal by microphages may be improved for smallerreporters. However, the reduced sensitivity available with smallerreporters must also be considered. One type of small up-convertinginorganic phosphor consists of rare earth ions in chelates. The use oflanthanide chelates as reporters has been developed for biologicalassays as described above. This prior use of lanthanide chelatesinvolved dowvn-conversion. That is, the emission light is at awavelength which is longer than the excitation wavelength. Rare earthchelates may be used as up-converting reporters through stepwiseexcitation such as shown in FIG. 5a, or in FIG. 5b (except that alllevels would be in the same ion). Energy transfer from a sensitizer ionto an activator ion cannot be used in the case of a single rare earthion. Chelates suitable for use as up-converting phosphors includeethylenediaminetetraacetic acid (EDTA), dipicolinic acid (DPA),diethylenetriaminetetraacetic acid (DTTA), diethylenetriaminepentaaceticacid (DTPA), tetraazacyclotetradecanetetraacetic acid (TETA), as well asantibiotics, natural chelating proteins, phthalocyanines, and cryptates.Methods for preparation of lanthanide chelates and their use inbiological assays are described in the literature (Mukkala et al. (1989)Anal. Biochem. 176: 319, Hemrnila et al. (1984)Anal. Biochem. 137: 335,Soini and Kojola (1983) Clin. Chem. 29: 65, Nonisotopic DNA ProbeTechniques (1992) Kricka (Ed.) Academic Press, New York, as well as thereferences on page 6 of this application). Up-conversion phosphorreporters can also consist of rare earth ions inside cage compounds suchas fullerene materials following the procedures described by Bethune etal. (1993) Nature 366: 123 and references therein.

Suitable ions for up-conversion in chelates include erbium, neodyrnium,thulium, holnium, and praseodymium. Other candidate ions include theother lanthanide elements, the actinide elements, and other metalelements. Stepwise excitation schemes suitable for up-conversion inlanthanide chelates are described in the literature on up-conversionlasers. Examples include up-conversion in erbium (Silversmith et al.(1986) J. Opt. Soc. Am. A3:128, and Macfarlane et al. (1989) Appl. PhysLett. 54:2301), neodymium (Macfarlane et al. (1988) Appl. Phsy. Lett.52:1300), thulium (Nguyen et al. (1989) Appl. Opt. 28:3553 and Allain etal. (1990a) Electron. Lett. 226:166), holmium (Allain et al. (1990b)Electron. Lett. 26:261), and praseodymium (Smart et al. (1991) Electron.Lett. 27:1307). Other up-conversion laser schemes that rely on energytransfer, energy pooling, cross relaxation, or avalanche absorption arenot appropriate for up-converting chelates because they rely on energytransfer between ions. These processes are described by Auzel (1973)Proc. IEEE 61:758 and Lenth and Macfarlane (March 1992) Optics andPhotonic News 3:8. Energy transfer can be efficient in a crystallinehost containing many rare earth ions, but not in a solution where theconcentration of ions is low and the phonon structure is lessconstrained. In certain cases, these schemes may not function as wellfor up-conversion in chelates. For example, certain of the schemes havebeen demonstrated using crystalline host materials at very lowtemperatures, and may not function as well at room temperature in achelate. Schemes that do not involve intermediate relaxation such asthat of Smart et al., have advantages in chelates because they can beexcited more effectively with pulsed sources. Higher peak powers can beobtained from diode lasers when they are operated in a pulsed mode. Thehigher peak powers lead to more efficient up-conversion due to thenonlinear dependence on excitation power.

Up-Converting Organic Dyes

Similar to the up-converting inorganic phosphor reporters we propose touse "molecular" labels whose fluorescence will be detected byoptoelectronic means. Infrared or red light is exciting theprobe-reporter complex bound to a target, after which light is emittedat shorter wavelengths with respect to the illuminating source. Thisup-converted light is free of scattered light from the source orautofluorescence by virtue of its higher energy. Furthermore,autofluorescence is greatly reduced by virtue of the excitation in theinfrared or red spectral range. The light source is a pump laser whosepump pulses are short in order to achieve high powers and low energy inorder to enable non-linear optical processes in the dye. The goal is toexcite the second excited singlet state (S₂) in a dye with a ps pulsefrom a tunable dye laser using two red or infrared photons. Afterpumping the S₂ state the dye relaxes within a few ps to the fluorescingstate (S₁) which can be detected by optoelectronic means. The goal ofreaching the S₂ slate using two photons enables one to take advantage ofthe increasing two-photon cross sections as one approaches the S2 stateusing two-photon absorption. The non-resonant two-photon absorptioncross sections are on the order of 10⁻⁴⁹ to 10⁻⁵⁰ cm⁴ s, whereas thecross sections corresponding to S₂ absorption are larger by two to threeorders of magnitude. A few specific examples will be mentioned: ingeneral cyanines, xanthenes, rhodamines, acridines and oxazines are wellsuited for this purpose. Blue dyes can also be used, but the excitationwavelength will be in the red. Rhodamine can be excited at 650 to 700 nmusing two photons, and fluorescence is expected around 555 nm. Many IRdyes such as IR-140, IR-132 and IR-125 can be excited at 1060 nm usingtwo photons of the Nd:YAG fundamental, and fluorescence is expected inthe 850 to 950 nm range. An example of a blue dye is BBQ excited at 480nm to reach the S₂ state at 240 nm, and fluorescence is expected at 390nm. Many of these dyes are only slightly soluble in aqueous solution andare either polar in nature (cyanines) or have polar subslituents.Depending on the nature of the probe, no or only minimal attachmentchemistry needs to be undertaken because of the abundance of functionalgroups on the dye chromophore. Several companies sell entire lines ofdyes: examples are KODAK, Exciton and Lambda Physik. The scientificfoundations of two-photon laser excitation in organic dye molecules havebeen treated in a few experimental papers: A. Penzkofer and W.Leupacher, Optical and Quantum Elecironics 19 (1987), 327-349; C. H.Chen and M. P. McCann, Optics Commun. 63 (1987), 335; J. P. Hermann andJ. Ducuing, Optics Commun. 6 (1972), 101; B. Foucault and J. P. Hermann,Optics Commun. 15 (1975), 412; Shichun Li and C. Y. She, Optica Acta 29(1982), 281-287; D. J. Bradley, M. H. R. Hutchinson and H. Koetser,Proc. R. Soc. Lond. A 329 (1972), 105-119.

Resonant Multiphoton Ionization

At very high laser intensities the up-converting organic dyes areinduced to absorb an additional exciting photon in the field of focussedlaser radiation. At those high laser intensities the Fluorescence issuppressed in favor of absorption of an additional photon. This processusually brings the organic dye molecules above the ionization limit insolution and they stabilize by emitting an electron into the solventshell. The result of this three-photon interaction is a molecular ionand an attached or solvated electron. When this charge separation istaking place in an electric field, the charges drift and generate avoltage that can be detected in an extremely sensitive manner. Thisamounts to the measurement of the transient conductivity in the solventsystem and is usually more sensitive than light detection. Thedisadvantage of this method is that it necessitates electrodes thatsense the moving charges. In that sense it is not as non-invasive amethod as light detection. On the other hand it bypasses the conversionof light into a photoelectric signal which represents an enormousadvantage. Every optical system has a restricted viewing angle thatreduces efficiency, whereas photoionization "senses" always close to100% of the charges generated. Effectively, the non-linear interactionof the laser field converts every excited organic dye molecule into anelectric pulse at sufficiently high field intensities that can beroutinely achieved using commercial laser sources. Specific examples arethe excitation of Rhodamine around 650 to 700 nm, or BBQ excitationaround 480 rm. Organic dyes absorbing in the red have to absorb twoadditional photons after being excited into S2 thus making the wholeprocess a four-photon excitation process, which is slower than athree-photon non-linear process. There may, however, be circumstanceswhere such a four-photon process is desirable.

Detection Apparatus

Detection and quantitation of inorganic up-converting phosphor(s) isgenerallyaccomplished by: (1) illuminating a sample suspected ofcontaining up-converting phosphors with electromagnetic radiation at anexcitation wavelength, and (2) detecting phosphorescent radiation at oneor more emission wavelength band(s). Illumination of the sample isproduced by exposing the sample to electromagnetic radiation produced byat least one excitation source. Various excitation sources may be used,including infrared laser diodes and incandescent filaments, as well asother suitable sources. Optical filters which have high transmissibilityin the excitation wavelength range(s) and low transmissibility in one ormore undesirable wavelength band(s) can be employed to filter outundesirable wavelengths from the source illumination. Undesirablewavelength ranges generally include those wavelengths that producedetectable sample autofluoresence and/or are within about 25-100 nm ofexcitation maxima wavelengths and thus are potential sources ofbackground noise from scattered excitation illumination. Excitationillumination may also be multiplexed and/or collimated; for example,beams of various discrete frequencies from multiple coherent sources(e.g., lasers) can be ccllimated and multiplexed using an array ofdichroic mirrors. In this way, samples containing multiple phosphorspecies having different excitation wavelength bands can be illuminatedat their excitation frequencies simultaneously. Illumination may becontinuous or pulsed, or may combine continuous wave (CW) and pulsedillumination where multiple illumination beams are multiplexed (e.g., apulsed beam is multiplexed with a CW beam), permitting signaldiscrimination between phosphorescence induced by the CW source andphosphorescence induced by the pulsed source, thus allowing thediscrimination of multiple phosphor species having similar emissionspectra but different excitation spectra. For example but notlimitation, commercially available gallium arsenide laser diodes can beused as an illumination source for providing near-infrared light.

The ability to use infrared excitation for stimulating up-convertingphosphors provides several advantages. First, inexpensive IR and near-IRdiode lasers can be used for sustained high-intensity excitationillumination, particularly in IR wavelength bands which are not absorbedby water. This level of high-intensity illumination would not besuitable for use with conventional labels, such as ordinary fluorescentdyes (e.g., FITC), since high-intensity UV or visible radiation producesextensive photobleaching of the label and, potentially, damage to thesample. The ability to use higher illumination intensities withoutphotobleaching or sample damage translates into larger potentialsignals, and hence more sensitive assays.

The compatibility of up-converting labels with the use of diode lasersas illumination sources provide other distinct advantages over lampsources and most other laser sources. First, diode laser intensity canbe modulated directly through modulation of the drive current. Thisallows modulation of the light for time-gated or phase-sensitivedetection techniques, which afford sensitivity enhancement without theuse of an additional modulator. Modulators require high-voltagecircuitry and expensive crystals, adding both cost and additional sizeto apparatus. The laser diode or light-emitting diode may be pulsedthrough direct current modulation. Second, laser illumination sourcesprovide illumination that is exceptionally monochromatic and can betightly focused on very small spot sizes, which provides advantages insignal-to-noise ratio and sensitivity due to reduced background lightoutside of the desired excitation spectral region and illuminatedvolume. A diode laser affords these significant advantages without theadditional expense and size of other conventional or laser sources.

Detection and quantitation of phosphorescent radiation from excitedup-converting phosphors can be accomplished by a variety of means.Various means of detecting phosphorescent emission(s) can be employed,including but not limited to: photomultiplier devices, avalanchephotodiode, charge-coupled devices (CCD), CID devices, photographic filmemulsion, photochemical reactions yielding detectable products, andvisual observation (e.g., fluorescent light microscopy). If thereporters are organic dyes, resonant multiphoton ionization can besensed using electrostatic position-sensitive detectors. Detection canemploy time-gated and/or frequency-gated light collection for rejectionof residual background noise. Time-gated detection is generallydesirable, as it provides a method for recording long-lived emission(s)after termination of illumination; thus, signal(s) attributable tophosphorescence or delayed fluorescence of up-converting phosphor isrecorded, while short-lived autofluoresence and scattered illuminationlight, if any, is rejected. Time-gated detection can be produced eitherby specified periodic mechanical blocking by a rotating blade (i.e.,mechanical chopper) or through electronic means wherein prompt signals(i.e., occurring within about 0.1 to 0.3 μs of termination ofillumination) are rejected (e.g., an electronic-controlled, solid-stateoptical shutter such as Pockel's or Kerr cells). Up-converting phosphorsand up-converting delayed fluorescent dyes typically have emissionlifetimes of approximately a few milliseconds (perhaps as much as 10 ms,but typically on the order of 1 ms), whereas background noise usuallydecays within about 100 nTs. Therefore, when using a pulsed excitationsource, it is generally desirable to use time-gated detection to rejectprompt signals.

Since up-converting phosphors are not subject to photobleaching, veryweak emitted phosphor signals can be collected and integrated over verylong detection times (continuous illumination or multiple pulsedillumination) to increase sensitivity of detection. Such timeintegration can be electronic or chemical (e.g., photographic film).When non-infrared photographic film is used as a means for detectingweak emitted signals, up-converting reporters provide the advantage ascompared to down-converting phosphors that the excitation source(s)typically provide illumination in a wavelength range (e.g., infrared andnear infrared) that does not produce significant exposure of the film(i.e., is similar to a darkroom safelight). Thus, up-convertingphosphors can be used as convenient ultrasensitive labels forimmunohistochemical staining and/or in situ hybridization in conjunctionwith fluorescence microscopy using an infrared source (e.g., a infraredlaser diode) and photographic film (e.g., Kodak Ektachrome) for signaland image detection of visible range luminescence (with or without aninfrared-blocking filter).

Instrumentation Overview

The basic purpose of the instrumentation is to expose the up-convertingphosphor particles of an assay sample to near-infrared (NIR) light andto measure the amount of visible light that is emitted.

FIG. 1 is an optical and electronic block diagram illustratingrepresentative apparatus 10 for performing diagnostics on a sample 15according to the present invention., The invention may be carried outwith one or a plurality of reporters. For purposes of illustration, theapparatus shows a system wherein two diagnostics are performed on asingle sample in which two phosphor reporters are used. The firstreporter has an excitation band centered at λ₁ and an emission bandcentered at λ₁ while the second reporter has respective excitation andemission bands centered at λ₂ and λ₂. Since the reporters of the presentinvention rely on multiphoton excitation, wavelengths λ₁ and λ₂ arelonger than wavelengths λ₁ and λ₂. The former are typically in the nearinfrared and the latter in the visible.

A pair of light sources 20(1) and 20(2), which may be laser diodes orlight-emitting diodes (LEDs), provide light at the desired excitationwavelengths, while respective detectors 22(1) and 22(2), which may bephotodiodes, detect light at the desired emission wavelengths. Theemitted radiation is related to the incident flux by a power law, soefficiency can be maximized by having the incident beam sharply focusedon the sample. To this end, light from the two sources is combined to asingle path by a suitable combination element 25, is focused to a smallregion by a lens or other focusing mechanism 27, and encounters thesample. Light emitted by the phosphor reporters is collected by a lens30, and components in the two emission bands are separated by a suitableseparation element 32 and directed to the respective detectors.

There are a number of possible regimes for driving the laser diodes anddetecting the emitted light in the different wavelength bands. This isshown generically as a control electronics block 35 communicating withthe laser diodes and detectors. The particular timing and othercharacteristics of the control electronics will be described below inconnection with specific embodiments.

There may be a plurality of reporters having distinct emission bands buta common excitation band. In such a case, the system would includemultiple detectors for a single laser diode. Similarly, there may be aplurality of reporters having distinct excitation bands but a commonemission band. In such a case, the system would include multiple laserdiodes for a single detector, and would use time multiplexing techniquesor the like to separate the wavelengths.

Light from the two sources is shown as being combined so as to befocused at a single location by a common focusing mechanism. This is notnecessary, even if it is desired to illuminate the same region of thesample. Similarly, the collection need not be via a single collectionmechanism. If it is necessary to preserve all the light, the combinationand separation elements can include a wavelength division multiplexerand a demultiplexer using dichroic filters. If loss can be tolerated,50% beam splitters and filters can be used.

The schematic shows the light passing through the sample and beingdetected in line. As a general matter, the emission from the phosphorreporters is generally isotropic, and it may be preferred to collectlight at an angle from the direction of the incident light to avoidbackground from the excitation source. However, since the excitation andthe emission bands are widely separated, such background is unlikely tobe an issue in most cases. Rather, other considerations may dictateother geometries. For example, it may be desired to detect lighttraveling back along the path of the incident radiation so that certainelements in the optical train are shared between the excitation and thedetection paths.

A typical type of instrument with shared elements is a microscope wherethe objective is used to focus the excitation radiation on the sampleand collect the emitted radiation. A potentially advantageous variationon such a configuration makes use of the phenomenon of optical trapping.In a situation where the reporter is bound to a small bead, it may bepossible to trap the bead in the region near the beam focus. The samesource, or a different source, can be used to excite the reporter. Theuse of an infrared diode laser to trap small particles is described inSato et al., "Optical trapping of small particles using a 1.3 μm compactInGaAsP laser," Optics Letters, Vol. 16, No. 5 (Mar. 1, 1991),incorporated herein by reference.

Specific Detection Techniques

As outlined above, multichannel detection uses optical devices such asfilters or dichroic beam. splitters where the emission bands of thephosphor reporters are sufficiently separated. Similarly, it was pointedout that multiple reporters having a common emission band could bedetected using electronic techniques. These electronic techniques willbe described below in conneticon with multiple sources. However, thetechniques will be first described in the context of a single channel.The techniques are useful in this context since there are sources ofbackground that are in the same wavelength range as the signal sought tobe measured.

FIG. 2A shows an apparatus for implementing phase sensitive detection inthe context of a single channel. Corresponding reference numerals areused for elements corresponding to those in earlier described figures.In this context, control electronics 35 comprises a waveform generator37 and a frequency mixer 40. Waveform generator 37 drives laser diode20(1) at a frequency f₁, and provides a signal at f₁ to the frequencymixer. The frequency mixer also receives the signal from detector 22(1)and a phase control input signal. This circuitry provides additionalbackground discrimination because the background has a much shorterlifetime than the signal sought to be measured (nanoseconds ormicroseconds compared to milliseconds). This causes the signal andbackground to have different phases (although they are both modulated atthe characteristic frequency of the waveform generator). For adiscussion of the lifetime-dependent phase shift, see Demtroder, LaserSpectroscopy, Springer-Verlag, New York, 1988, fop. 557-559,incorporated herein by reference). The phase input signal is controlledto maximize the signal and discriminate against the background. Thisbackground discrimination differs from that typical for phase sensitivedetection where the signal is modulated and the background is not.Discrimination against unmodulated background is also beneficial here,leading to two types of discrimination.

Because the signal relies on two-photon excitation, it is possible touse two modulated laser diodes and to detect the signal at the sum ordifference of the modulation frequencies. FIG. 2B shows such anarrangement where first and second laser diodes 20(1) and 20(1)'(emitting at the same wavelength λ₁, or possibly different wavelengths)are modulated by signals from waveform generators 37a and 37b operatingat respective frequencies f_(a) and f_(b). The waveform generator outputsignals are communicated to a first frequency mixer 42, and a signal atf_(a) ±f_(b) is communicated to a second frequency mixer 45. The signalfrom detector 22(1) and a phase input signal are also communicated tofrequency mixer 45.

FIG. 3 shows apparatus for performing gated detection. Since thebackground is shorter-lived than the signal, delaying the detectionallows improved discrimination. To this end, the laser diode is drivenby a pulse generator 50, a delayed output of which is used to enable agated integrator or other gated analyzer 55.

FIG. 4 shows an apparatus for performing diagnostics on a sample usingfirst and second, reporters having excitation bands centered at λ₁ andA₂, and having overlapping emission bands near λ₃. The sample isirradiated by light from laser diodes 20(1) and 20(2) as discussed abovein connection with FIG. 1. First and second waveform generators 37(1)and 37(2) drive the laser diodes at respective frequencies f₁ and f₂,and further provide signals at f₁ and f₂ to respective frequency mixers60(1) and 60(2). The signal from detector 22(3) is communicated to bothfrequency mixers, which also receive respective phase input signals.Thus, frequency mixer 60(1) provides an output signal corresponding tothe amount of emitted light modulated at frequency f₁, which provides ameasure of the presence of the first reporter in the sample. Similarly,frequency mixer 60(2) provides an output signal corresponding to theamount of emitted light modulated at frequency f₂, which provides ameasure of the presence of the second reporter in the sample.

The use of two different wavelengths was discussed above in the contextof two reporters having different excitation bands. However, thediscussion is germane to a single reporter situation as well. Since theexcitation is a two-photon process, there is no requirement that the twophotons have the same energy. Rather, it is only necessary that thetotal energy of the two photons fall within the excitation band. Thus,since it is relatively straightforward and inexpensive to providedifferent wavelengths with laser diodes, there are more possiblecombinations, i.e., more possible choices of total excitation energy.This allows more latitude in the choice of rare earth ions forup-converters since the excitation steps need not rely on energytransfer coincidences involving a single photon energy. Further, it maybe possible to achieve direct stepwise excitation of the emitting ion(the erbium ion in the example outlined above) without using energytransfer from another absorbing ion (the ytterbium ion in the example)while taking advantage of resonant enhancement of intermediate levels.Additionally, the use of different wavelengths for a single reporter canprovide additional options for excitation-dependent multiplexing andbackground discrimination techniques.

Multiple wavelength excitation of a single phosphor may occur in anumber of ways, as shown in FIGS. 5A through 5C. Two lasers may causestepwise excitation of a single ion, as shown in FIG. 5A. A first laserstimulates excitation from level 1 to level 2, and a second laserstimulates excitation from level 2 to level 3, at which level emissionoccurs. Single ion excitation can also occur using energy transfer asshown in FIG. 5B. In this case, a first laser stimulates excitation fromlevel 1 to level 2, energy transfer occurs from level 2 to level 3, anda second laser stimulates excitation from level 3 to level 4. In avariation of the latter process, levels 1 and 2 can be in a first ion(i.e., a sensitizer ion) and levels 3 and 4 in a second ion (i.e.,activator ion) as shown in FIG. 5C.

In a stepwise excitation scheme shown in FIG. 5A, energy transfer is notrequired, and thus information on the polarization of the excitationlasers may be preserved and cause polarization of the emitted radiation.In this case, depolarization of the light may allow for enhanceddiscrimination between signal and background noise.

For the multi-ion multi-laser excitation scheme shown in FIG. 5C, theremay be several phosphors that share a common excitation wavelength. Inthis case, discrimination between different phosphors may be performedon the basis of different emission wavelengths and/or throughtime-gated, frequency-modulated, and/or phase-sensitive detectionutilizing modulation of the excitation wavelength(s).

Specific Instrument Embodiments

FIG. 6 is a schematic view snowing the optical train of a particularembodiment of apparatus for carrying out the present invention on asample using a hand-held probe. This embodiment takes the form of aminiaturized instrument comprising a housing 75 (shown in phantom), ahand-held probe 80, with a fiber optic connecting cable 82. The opticaland electronics components are located within the housing. For purposesof illustration, the optical components of a 3-channel system are shown.The sample may contain up to three reporters having distinct emissionbands, for example, in the blue, green, and red portions of the visiblespectrum. It is also assumed that the reporters have distinct excitationbands in the near infrared.

The output beams from three laser diodes 85a-c are communicated throughgraded index (GRIN) lenses 87a-c, focused onto the ends of respectivefiber segments 88a-c and coupled into a single fiber 90 by a directionalcoupler 92 or other suitable device. The light emerging from the end offiber 90 is collimated by a GRIN lens 95, passes through a dichroic beamsplitter 97, and is refocused by a GRIN lens 100 onto the end of fiberoptic cable 82. The beam splitter is assumed to pass the infraredradiation from the laser diodes but reflect visible light.

Hand-held probe 80 includes a handpiece 102, an internal GRIN lens 105,and a frustoconical alignment tip 110. The light emerging from fiber 82is focused by GRIN lens 105 at a focus point 115 that is slightly beyondalignment tip 110. The alignment tip is brought into proximity with thetest tube holding the sample so that focus point 115 is in the sample.It is assumed that the test tube is transmissive to the laser radiation.

A portion of the light emanating from the region of focus point 115 inthe sample is collected by GRIN lens 105, focused into fiber 82,collimated by GRIN lens 100, and reflected at dichroic beam splitter 97.This light may contain wavelengths in up to the three emission bands.

Optical filters 120a-c direct the particular components to respectivephotodetectors 125a-c. A particular filter arrangement is shown whereeach filter reflects light in a respective emission band, but otherarrangements would be used if, for example, one or more of the filterswere bandpass filters for the emission bands.

The control electronics are not shown, but could incorporate thetime-multiplexed or heterodyne techniques discussed above. Suchtechniques would be necessary, for example, if the emission bands werenot distinct.

FIG. 7A is a schematic of an embodiment of the invention in which acharge coupled device (CCD) imaging array 150 is used as a detector incombination with a two dimensional array 152 of peptides or otherbiologically active species deposited on a glass or plastic substrate.

The CCD array has a number of individually addressable photosensitivedetector elements 155 with an overlying passivation layer 157 while thepeptide array has a number of individual binding sites 160. The probecontaining the phosphor would be reaction specific to one or more of theelements in this peptide array and would therefore become physicallyattached to those elements and only those elements. The peptide array isshown as having a one-to-one geometric relation to the imaging array inwhich one pixel corresponds to each element in the peptide array.However, it is also possible to have larger peptide elements that covera group of detector elements should such be necessary.

Various of the techniques described above can be used to enable thedetector array to distinguish the emissions of the phosphor from theinfrared laser stimulation. First, it is possible to use a phosphor thatresponds to IR stimulation beyond the sensitivity range of the detectorarray. An example of such a phosphor would be Gadolinium oxysulfide: 10%Erbium. This phosphor is stimulated by 1.5-micron radiation and emits at960 nm and 520 nm. The detector array is insensitive to 1.5-micronradiation but is sensitive to the up-converted radiation.

Further, since the phosphor emission is relatively slow in rise and falltime it could be time resolved from a pulsed laser stimulation source bythe CCD detector array. The decay time for the up conversion process isa variable dependent on the particular emitting transition and thephosphor host; however, it is normally in the range 500 μis seconds to10 ms. This is very slow compared to the laser excitation pulse and thecapability of the detector array. it The techniques for fabricating theCCD array are well-known since CCD imaging arrays have been commerciallyavailable for many years. A variety of such devices can be obtained fromDavid Sarnoff Research Center, Princeton, N.J.

The techniques for fabricating the peptide array are described in apaper by Fodor et al., "Light-Directed, Spatially Addressable ParallelChemical Synthesis," Science, Vol. 251, pp. 767-773 (Feb. 15, 1991),incorporated herein by reference. The particular array discrete contains1024 discrete elements in a 1.28 cm×1.28 cm area.

The embodiment of FIG. 7A shows the peptide array in intimate contactwith the CCD array. Indeed it may be possible to deposit the peptidesdirectly on the passivation layer without a separate substrate. However,there may be situations where spatially separated arrays are preferredFIG. 7B shows an embodiment where the peptide array and the CCD arrayare separated. An array of lenses 165 collect the light from respectivebinding sites and focus it on respective detector elements. Thisarrangement facilitates the use of filters to the extent that othertechniques for rejecting the excitation radiation are not used.

Optical trapping may be used to transiently immobilize a sample particlefor determination of the presence or absence of phosphor on theparticle. Conveniently, the wavelength range used to trap sampleparticles may be essentially identical to an excitation wavelength rangefor the up-converting phosphor(s) selected, so that optical trapping andexcitation illumination is performed with the same source. FIG. 3 showsa block diagram of an apparatus used for single-beam gradient forcetrapping of small particles.

FIG. 26 is a block diagram of one embodiment of apparatus for carryingout the present invention on a sample using a microscope. In thisembodiment a standard microscope is modified to accept infrared scanningoptics and image processing electronics. A suitable microscope formodification is the Zeiss model CLSM-10.

The microscope is fitted with a HeNe laser A1 for visible imaging and anargon laser A2 for both visible and UV imaging. Both lasers are mountedinternally and are individually selectable through a series of motorizedshutters A3. The upconverting phosphors are excited with an externallymounted IR laser diode. In the preferred embodiment, two IR laser diodesA4 and A5, operating at two different IR wavelengths, are coupled to themicroscope thereby allowing multiple phosphor reporters to beidentified. Laser diodes A4 and A5 are individually selectable usingmotorized shutters A6. When an IR beam is selected, it is routed throughthe microscope's galvanometrically controlled scanning mirrors A7 whichscan the beam in a raster fashion. The beam passes through the objectivelens (not shown) onto a sample A8 and is reflected back through theobjective lens to a set of galvanometrically controlled receivingmirrors A7. Receiving mirrors A7 reflect the light onto pinhole opticsA9. If the confocal mode is selected, pinhole A9 limits the detectedimage to the light collected from the focal plane. The light is imagedon a photomultiplier tube (PMT) A10. The thickness of the focal plane isproportional to the size of the pinhole. The scanning speed is chosensuch that sufficient signal intensity is received at the PMT A10. In thepreferred embodiment of this apparatus, a 20 micrometer diameter pinholeis used which results in a depth of field of about 1 micrometer. If theconfocal mode is not selected, the beam is deflected around pinholeoptics A9 directly to PMT A10.

Once the optical signal is converted into an electronic one, a standard,composite video signal can be developed and displayed as an image on atelevision monitor A11. The image can be manipulated and enhancedthrough standard image processing software. in the preferred embodimentof this apparatus the software runs on an IBM 486 PC A12. The softwarecan be used to perform averaging, filtering, edge detection andoverlaying the images received from each of the different light sources.

In the confocal mode, it is possible to reconstruct a 3 dimensional viewof sample A8. The reconstruction is formed by stepping through sample A8at small intervals, making an image of the sample at each interval. Themultiple sequential images are transferred to an external graphicsmachine (not shown) for reconstruction of the sample in 3 dimensions.These 3-D images can then be rotated to give different perspectives ofthe data sets, leading to a better understanding of the samples.

FIG. 27 is a block diagram of a microtiter plate reader for use with thepresent invention. Within a light-tight test chamber B1 is a near IRlaser excitation source B2, a photomultiplier tube (PMT) detector B3,and a sample assay plate B4. In the preferred embodiment of thisapparatus, assay plate B4 is a Terasaki HLA plate. This plate ispreferred due to its small tapered sample wells which tend toconcentrate the sample material into a relatively small target area. Thetarget area in this configuration is still larger than the diameter ofthe laser beam. Furthermore, it is possible that the distribution of theassay material across the bottom of the well is not even. Because ofthese two factors, simply aiming the laser at the center of the bottomwell surface is unlikely to provide accurate readings. There are severalapproaches that can be used to circumvent this problem. The firstapproach is to defocus the laser beam sufficiently to allow a largeramount of the target area to be interrogated. However, depending uponthe output of laser B2, defocussing the beam may lower the sensitivityof the apparatus to an unacceptable level. Another approach is to rasterscan the laser beam across the bottom of target well. A third andpreferred approach is to simply automate the scanning and datacollection system.

Light from laser B2 passes through a filter B5 and is focussed by a lensB6 onto an individual sample well of assay plate B4. Plate B4 is mountedon a pair of translators B7 which allow positioning in the horizontaland vertical directions. In the present configuration translators B7allow approximately 2.5 centimeters of travel; sufficient to address 3sample wells in each direction. Translators B7 are controlled by an x-ycontroller B8. Controller B8 allows for either manual or computerizedcontrol.

A sample well on plate B4, when containing upconverting phosphors, willemit visible light which is collected by a lens B9, passed through afilter B10, and focussed through a lens B11 and a shutter B12 onto PMTB3. PMT B3 outputs a current which is measured by a picoammeter B13. ThePMT signal is proportional to the phosphor emission intensity. ShutterB12, controlled by a shutter driver B14, provides exposure protection toPMT B3, thereby preventing damage which may result from exposure to veryintense light sources. Furthermore, overexposure of PMT B3 to lightcauses high dark currents which require several hours to decrease. PMTB3 is cooled for lower dark current and noise. Associated with the PMTcooler is a water-cooled power supply B15. A power supply B16 supplieshigh voltage to PMT B3.

When the apparatus is operated in a computerized mode, a computer B17regulates controller B8 through an interface box B18. Picoammeter B13can also be connected to computer B17, thereby allowing automated dataacquisition to be performed. The data acquisition procedure movestranslator B7 in the x direction to a first position at which location aspecified number of current readings are taken and the average iscalculated. Translator B7 then movres sample B4 a predetermined distancein the x direction to a new location where new data is collected. Duringthis process, the data is plotted in order to provide the user with animmediate visual evaluation. After the scan is completed, the data canbe saved or further data processing can be performed. FIG. 28 is anillustration of the data for upconverting phosphors in three test wells.

FIG. 29 is a schematic view of a second embodiment of a hand-held probefor carrying out the present invention. This embodiment is comprised ofa housing Dl and a capillary wick D2. Within housing Dl is a diodeexcitation laser D3, a lens assembly D4, a photodiode detector D5, ad abattery supply D6. A display D7 mounted to one surface of housing Dlcommunicates the results of the test to the user. In the preferredembodiment, laser D3 operates in the 960-980 nanometer range.

In use, wick D2 wicks up a portion of a sample fluid D8 which issuspected of containing the target antigens. Target antigens bind to theantibodies present at a capture surface D9. Capture surface D9 ispositioned at the focal point of source D3. The target antigens can belabeled with phosphor-antibody conjugates either before or aftercapture. In the preferred embodiment wick D2 is formed of glass. In thisconfiguration capture surface D9 is prepared simply by filling theinside of the capillary with a bubble containing the antibodies ofinterest. By silanizing the inner surface with organofunctional silanes,conventional chemistries can be used to covalently link the antibodiesor other biological macromolecule(s) to the inner tube wall at the siteof the liquid bubble. The surface energy of the capillary is also easyto modify by silanization, which will help prevent nonspecific reagentand antigen adherence to the walls of the tube.

In the preferred embodiment of this apparatus, the lower portion of wickD2 is impregnated with upconverting phosphors that are conjugated to thetarget analytes or a crossreactive epitope for the capture probe. Inuse, the phosphor conjugates chromatograph towards capture surface D9 assample fluid D8 is drawn up wick D2. As phosphors accumulate at capturesurface D9, they will begin to emit visible light upon excitation bydiode laser D3. The visible light emitted by the phosphors is detectedby detector D5. The output of detector D5 is displayed on display D7.The amount of upconverted light reaching the detector is directlyproportional to the concentration of labeled target antigen captured atthe capture surface.

The apparatus of FIG. 29 can be designed to simultaneously detect morethan one target antigen. FIG. 30 illustrates a three channelconfiguration using interference filters. In this configurationcapillary wick D2 is placed at the focus of a small parabolic reflectorD10 capable of collecting approximately half of the emittedphosphorescence. The beam from diode laser D3 is directed onto capillarywick D2 at capture surface D9 along a direction perpendicular to theoptical axis of reflector D10. Phosphorescent light from capture surfaceD9 is collected and collimated by mirror D10, directed through a notchfilter D11 to reject the pump light, and onto three detectors D5 usingthree dichroic beam splitters D12. The reflectance bands of dichroicbeamsplitters D12 are matched to the emission bands of the threephosphors used in the detection process.

In an alternate embodiment of this apparatus, dichroic beamsplitters D12could be replaced with three bandpass filters used in the transmissionmode. By placing the three filters on a rotation wheel, a singledetector D5 could be used. Another alternative is to use a diffractiongrating and a linear detector array to obtain an actual emissionspectrum.

FIG. 31A is an illustration of an embodiment of the invention in which adiode laser array F1 and a detector array F2 are combined in a singledevice. In the preferred embodiment, arrays F1 and F2 are fabricated ona pair of silicon chips F3 with array dimensions of approximately 1square centimeter. FIG. 31B is a detailed view of a small section of thedevice shown in FIG. 31A. Overlaying detector array F2 is a polymer filmF4 of approximately 10 to 25 micrometers thickness which is used as thecapture surface. Arrays F1 and F2 are separated by a spacer F5. Array F1is comprised of Fabrey-Perot diode lasers, preferably tuned to 980nanometers. Lasers of this type are easily fabricated in gridded arraypatterns using conventional photolithography techniques. Each individuallaser in array F1 has a columnar beam designed to strike only theadjacent portion of capture surface F4. The required power density ofthe individual lasers is dependent upon the efficiencies of thephosphors being used as well as the required detection efficiency. Thedetectors comprising array F2 are chosen to have an extremely lowsensitivity in the ,wavelength region in which laser array F1 operates.If additional discrimination between the excitation and emissionwavelengths is required, a cutoff filter can be used, preferablyincorporated directly into capture surface F3.

Up-converting phosphors F6 are conjugated by any of a variety ofconventional biochemical crosslinking chemistries to antibody, nucleicacid probes, or other biological macromolecules (e.g., carbohydrates,lectins, streptavidin, MEC complexes), as well as to biological orchemical antigens (F7). Bonded to overlay F3 is a grid array F8 ofcomplementary probes or antigens which are bound to capture surface F3using the same crosslinking chemistries. In use, a sample fluid F9 flowsbetween arrays F1 and F2, target probes or antigens are captured by gridarray F8 and excited by laser array F1, and the emissions detected bydetector array F2.

Typically, the upconverting phosphors to be used with this apparatus areapproximately 0.1 to 0.5 micrometers. Since the size of the individualphosphor particles is of the order of the excitation wavelength, thepower of the emission from the phosphors can be approximated by:

    P.sub.em =fND.sup.3.4 I.sub.ex.sup.2

where f is the phosphorescence efficiency (generally less than or equalto 10⁻¹⁷ cm⁴ W⁻¹ μm⁻³,4 particle⁻¹), N is the number of phosphorparticles in the light path, D is the diameter of the phosphorparticles, and I_(ex) is the power density of the excitation source.

Since the emitted power scales as the square of the excitationintensity, diagnostics using upconverting phosphors perform better in amicroassay format. Assuming a constant power output from the excitationsource, the excitation power density increases proportionally with thedecrease in detection area, and the number of phosphor particles in thelight path decreases linearly with a decrease in the detection area.Since the power of the light emitted from the phosphors scales with thesquare of the excitation power density, but linearly with the number ofphosphors, P_(em) will increase in inverse proportion to the detectionarea. Therefore, a 100×100 array will actually be 100 times moresensitive than a 10×10 array.

Fluorescence-activated Cell Sorting

The up-converting phosphors described herein can be used asphosphorescent labels in fluorescent cell sorting by flow cytometry.Unlike conventional fluorescent dyes, up-converting phosphors possessthe distinct advantage of not requiring excitation illumination inwavelength ranges (e.g., UV) that damage genetic material and cells.Typically, up-converting phosphor labels are attached to a bindingreagent, such as an antibody, that binds with high affinity andspecificity to a cell surface protein present on a subset of cells in apopulation of cells in suspension. The phosphor-labeled bindingcomponent is contacted with the cell suspension under bindingconditions, so that cells having the cell surface protein bind to thelabeled binding reagent, whereas cells lacking the cell surface proteindo not substantially bind to the labeled binding reagent. The suspendedcells are passed across a sample detector under conditions wherein onlyabout one individual cell is present in a sample detection zone at atime. A source, typically an IR laser, illuminates each cell and adetector, typically a photomultiplier or photodiode, detects emittedradiation. The detector controls gating of the cell in the detectionzone into one of a plurality of sample collection regions on the basisof the signal(s) detected. A general description of FACS apparatus andmethods in provided in U.S. Pat. Nos. 4,172,227; 4,347,935; 4,661,913;4,667,830; 5,093,234; 5,094,940; and 5,144,224, incorporated herein byreference. It is preferred that up-converting phosphors used as labelsfor FACS methods have excitation range(s) (and preferably also emissionrange(s)) which do not damage cells or genetic material; generally,radiation in the far red, and infrared ranges are preferred forexcitation. It is believed that radiation in the range of 200 nm to 400nm should be avoided, where possible, and the wavelength range 760 nm to765 nm may be avoided in applications where maintenance of viable cellsis desired.

Additional Variations

There are several apparatus design issues relating to the uniqueexcitation and emission characteristics of upconverting phosphors whichmust be considered when using up-converting phosphors with flowcytometry. The first issue is the time required to reach maximumemission intensity. Since upconversion is a two photon process,upconverting phosphor emission is time delayed approximately 100microseconds. The phosphor must remain within the excitation beam forthis period of time regardless of the flow rate. Therefore given a flowrate between 1 and 10 meters per second with a channel width of 70 to200 micrometers, the length of the excitation beam must be between 100and 1000 micrometers. Given that the phosphor emissions saturate at anexcitation intensity of about 200 watts per square centimeter, the lasersource typically must have a power between 0.01 and 400 milliwatts toachieve phosphor saturation. This implies that multiple laser diodes maybe required to obtain maximum phosphorescence at the fastest flow rates.

Another design issue is that associated with the detector. Since thereis a considerable separation between the excitation and emissionwavelengths of the upconverting phosphors, detection can be performedusing a photomultipler tube (PMT), a photodiode, or a CCD array. Thephosphorescence decay time is long, with a decay half life ofapproximately 300 microseconds. The most sensitive method of detectionis to integrate the signal measured by the PMT. However, 99 percentdetection of the available phosphorescent signal requires that thephosphor remain in the sight path of the detector for 5 times thephosphorescence decay half-life (i.e., 1.5 milliseconds). Assuming aflow rate of 10 meters per second and a channel width of 200micrometers, the PMT must be able to detect over a path length of 1.5centimeters. This path length is also the required spacing between cellsflowing through the cytometer, implying a maximum count rate of 667cells per second. It is, however, possible to sacrifice some detectionsensitivity by reducing the detection path length, at least to thatrequired to attain steady-state emission from the phosphors. As long asa steady-state emission peak is reached by the phosphor in theexcitation window, the peak signal received by the PMT should bedirectly proportional to the concentration of phosphors present. Thenonphotobleaching property of the phosphors makes this form of detectionpossible. The loss in detection sensitivity corresponding to a 0.1centimeter path length (versus a 1.5 centimeter path length) isapproximately a factor of 3. Triggering the emission detector can beaccomplished by observing the light scattered by the cell as it passesthrough the excitation source.

In environments where absorption of the up-converted phosphor radiationis high, the phosphor microparticles are coated with a fluorescent dyeor combination of dyes, in selected proportions, which absorb at theup-converted frequency and subsequently re-radiate at other wavelengths.Because the single-photon absorption cross-sections for these fluors aretypically very high, only a thin layer is required for completeabsorption of the phosphor emission. This coat particle may then beencapsulated and coated in a suitable antigen or antibody receptor (e.g.microparticle). An example of this layering is depicted schematically inFIG. 9. There exists a wide variety of fluorescent dyes with strongabsorption transitions in the visible, and their emission covers thevisible range and extends into the infrared. Most have fluorescentefficiencies of 10% or more. In this manner, the emission wavelengthsmay be custom-tailored to pass through the particle's environment, andoptical interference filters may again used to distinguish betweenexcitation and emission wavelengths. If a relatively large wavelength"window" in the test medium exists, then the variety of emissionwavelengths which may be coated on a single type of phosphor is limitedonly by the number of available dyes and dye combinations.Discrimination between various reporters is then readily carried outusing the spectroscopic and multiplexing techniques described herein.Thus, the number of probereporter "fingerprints" which may be devisedand used in a heterogenous mixture of multiple targets is virtuallyunlimited.

The principles described above may also be adapted to drivingspecies-specific photocatalytic and photochemical reactions. In additionto spectroscopic selection, the long emission decay times of thephosphors permit relatively slow reactions or series of reactions totake place within the emission following photoexposure. This isespecially useful when the phosphor-[catalyst or reactant] conjugateenters an environment through which the excitation wavelength cannotpenetrate. This slow release also increases the probability that moretargets will interact with the particle.

The unique decay rates of phosphor particles allow dynamic studies aswell. In a system where continuous exposure to the excitation source isnot possible, or is invasive and thereby undesirable, pulsed excitationfollowed by delayed fluorescence detection is necessary. After thephosphor reporter has been photoexcited, the subsequent emission fromthe phosphor or phosphor/dye conjugate particle lasts typically about amillisecond. In a dynamic environment, such as a static or flowingsystem with moving targets, the particle will emit a characteristicallydecaying intensity of light as it travels relative to the excitationdetection apparatus. Combined with imaging optics appropriate to thescale of the system and the velocities within the system, a CCDphotoelectric sensor array will be used to detect the particle orparticles movement across the array's field of view. The delayedemission of the phosphors, which is a well-characterized function oftime, makes possible the dynamic tracking of individual particle'spositions, directions and velocities, and optionally calculation ofparticle size, density, and hydrodynamic conformation. As a particlemoves, it exposes more elements of the array, but with every-decreasingintensity. The more elements it exposes over a certain fraction of itsdecay time, the faster it is moving. Therefore, the integrated intensitypattern of a particle's emission "track" collected by the array isdirectly related to the velocity of the particle. The particles may berefreshed again at any time by the pulsed or chopped CW excitationsource. FIG. 10 illustrates this scheme. Although only a depiction of"side-on" excitation and detection is shown, both side-on and end-ondetection and excitation arrangements, or combinations, are possible.Reduction of the CCD array intensity information by computer analysiswill allow near-real time tracking of the particles in a dynamicallyevolving or living systems. Data analysis and reduction performed by thecomputer would include a convolution of the intrinsic decay of thephosphor emission, the number of pixels illuminated and their signallevel, the orientation of the decaying signal on the array, and theintensity contributions from a blur circle from particles moving in andout of the focal plane of the array. In an end-on flow detectionarrangement, the size of the blur circle would relate directly to howquickly the particle moves out of focus, thereby allowing the velocityof the particle to be determined. One possible application would bemonitoring the chemistry and kinetics in a reaction column,alternatively, the application of this method to flow cytometry maypermit the resolution of cells on the basis of hydrodynamic properties(size, shape, density). The method may also be useful for in vivodiagnostic applications (e.g., blood perfusion rate).

Up-converting phosphor labels may also be used to sense the temperaturein the region at which the up-converting phosphor label is bound.Up-converting phosphor temperature measurement methods are described inBerthou H. and Jorgensen C. K. (October, 1990) Optics Lett. 15(19):1100, incorporated herein by reference.

Matched Label Methods

As described above, photophysical catalysis, diagnostic assays and othersampling techniques can take advantage of matching the absorptionspectrum of a luminescent label with the emission spectrum of anup-converting label. The matching of an up-converting label with aluminescent label, i.e., a label which emits radiation upon absorptionof energy, represents a step beyond traditional photophysical catalysisand assay methods.

The term "matched labels" refers to two or more labels where at leastone label absorbs excitation radiation and emits energy which in turnexcites another label and causes it to emit emission radiation. In amatched label pair, an excitation label, such as an up-convertingphosphor, absorbs the excitation radiation and emits energy which inturn excites an emission label, such as a luminescent label. Theemission label, in a matched label pair, is excited by the energyemitted from the excitation label, and emits emission radiation whichmay be detected in an assay or used in photophysical catalysis orphotodynamic therapy. Additionally, a matched label pair may have one ormore transfer labels in addition to the excitation label and theemission label. Transfer labels are luminescent labels which absorb andrelay the excitation energy from the excitation label to an emissionlabel, causing the emission label to excite and emit radiation.

The matched labels of the invention are convenient and reliable forphotophysical catalysis and for detecting the presence of lowconcentrations of analytes. By employing matching up-converting andluminescent labels, it is possible to detect target analytes withnondestructive visible and infrared light, thus, producing little or nobackground interference. Furthermore, catalysis, detection anddiscrimination of multiple target analytes may be achieved with matchedlabels.

The matched labels may be employed in any traditional assay orphotophysical catalysis method. For example, in matched label assays, asample suspected of containing a target analyte is contacted withmatching up-converting and luminescent labeled probes. The up-convertinglabeled probes and luminescent labeled probes are capable ofspecifically binding the target analyte and forming labeled probe-targetcomplexes. Having bound the analyte to the labeled probes, the sample isilluminated with illumination energy to excite the excitation label. Thesample is then observed for emission radiation from the emission label.The detection of an emission from the emission label verifies thepresence of the target analyte since the emission label generally onlyabsorbs the energy caused by excitation label emissions when the matchedlabel s are attached to the target analyte. The apparatus used toilluminate the sample with an illumination wavelength and to detect anemission wavelength from the emission label is the same as describedabove.

The matched label assays may be carried out as homogeneous,heterogeneous or competitive assays. However, when using matchinglabeled probes in a homogeneous assay, it is preferred that the matchinglabels are attached to two probes to form distinct labeled-probe pairswhich bind to the target analyte. The probe may be the same or differentprobe material. For example, an up-converting label may be attached to afirst antigen probe which binds to one epitope site on the analyte whilethe matched luminescent label is bound to a different antigen probewhich binds to another epitope site on the same analyte.

Matched label photophysical catalysis can be conducted in a mannersimilar to the matched label assays described above except that theenergy from the emission label may be used to produce localized intenseelectromagnetic radiation for purposes other than detection. Forexample, the energy from the emission label may be used to bring about amodification in the target analyte or in materials adjacent to thetarget analyte. Another example of photophysical catalysis involves theuse of an up-converting label which excites a luminescent label causingit to emit its energy to create a reactive chemical species.

Both the matched label photophysical catalysis and assays of theinvention may employ a multitude of different matched labels and/ordifferent probes within a single sample. By varying the labels andprobes a wide variety of analytes may undergo photophysical catalysis orbe detected within a single sample. For example, one set of matchedlabels may contain a luminescent label as the excitation label and anup-converting label as its matched emission label while another matchedset in the same sample could contain an up-converting label as theexcitation label and a luminescent label as the emission label.

The following four embodiments outline the ability to conduct multipleassays within a single sample:

(1) In a single sample, the matching up-converting and luminescentlabels may be separately attached to two distinct probes of the same ordifferent material such that the matching labels are attached to probeswhich bind to the same analyte or overlapping sets of analytes. Forexample, a particular up-converting labeled probe may have affinity forone or several analytes and the matched luminescent labeled probe mayhave affinity for a portion of those analytes as well as other analytes.Thus, use of matched labeled-probe pairs allows for the detection ofanalytes which bind to both of the labeled probes or, in other words, anoverlapping set of analytes for the labeled probes.

When employing up-converting and luminescent labels attached to twodistinct probes, the excitation label preferably does not excite anemission label unless both of the distinct probes are in closeproximity, e.g., are bound to the same analyte. Thus, different targetanalytes may be detected in an assay. For example, different matchedlabel pairs may be attached to two distinct probes which bind to twodifferent target analytes. Then, for an assay method, by exciting theexcitation label and observing for emission radiations from the emissionlabels, the presence of one or both of the two different target analytesmay be detected within a single sample.

(2) In another embodiment, distinct sets of matched excitation andemission labels may be employed. For example, two different sets ofmatched labels each having distinct excitation labels and distinctemission labels may be used in a single sample. For example, in an assaymethod, by exciting the first excitation label and observing emissionradiation from its matched label, a first analyte may be detected.Additionally, in the same sample, a second analyte may be detected byexciting the second excitation label and observing emission radiationfrom its matched label.

(3) In another embodiment, a probe having one excitation label may bematched with at least two probes having distinct emission labels. Thus,in an assay, different target analytes may be detected within a singlesample by exciting the excitation label and then observing the differentemission radiations from the distinct emission labels.

(4) Another embodiment of the invention involves using matched labelshaving one or more emission labels which emit a detectable emissionradiation and at least two distinct excitation labels. For example, inan assay on a single sample, different target analytes may be detectedby separately exciting the distinct excitation labels and observing theemission radiation from the emission labels.

Of course, the various combinations of matched label pairs discussedabove may be employed with different probes to detect or, as discussedabove for photophysical catalysis or photodynamic therapy, effect amultitude of different target analytes within a single sample.Furthermore, these multiplexing embodiments may be carried outsimultaneously or sequentially upon a single sample.

Matched labels may also be used in other assay formats, (for example acompetitive assay), where a first labeled probe of a matched labeledprobe pair binds with a target analyte to form an initial labeledprobe-analyte complex which may be separated from the original sampleand then contacted with the matching labeled probe of the labeled probepair. By contacting the initial probe-analyte with the matching labeledprobe, the target analyte may be displaced such that the matchinglabeled probes may bind together. Illuminating the resulting sampleexcites the excitation label of the matched label pair. Emission fromthe emission label then indicates the presence of the target analyte.Alternatively, a target analyte may displace a labeled probe from abound matched label probe pair. In this situation, illumination and thedecrease or complete absence of emission radiation indicates thepresence of the target analyte.

In a preferred embodiment, matched labels involve the matching ofluminescent labels and up-converting labels. As described above,luminescent labels emit radiation, such as light, upon absorption ofenergy. The luminescent labels and up-converting labels may bephosphorescent, fluorescent, or chemilumunescent and generally emitlight having a wavelength in the infrared, visible or ultravioletregions.

The up-converting labels of the invention include up-convertinginorganic phosphors, dyes, chelates and non-linear optical compounds.Examples of up-converting dyes and inorganic phosphors suitable for usein the invention include, but are not limited to, those described above.Examples of up-converting non-linear optical compounds include, but arenot limited to,4-(p-nitrolphenyl)-3,4,dihydro-pyrazo[c]-benzo[b]morpholine (NDPB),1-o-tolyl-3-p-nitrophenyl-2-pyrazoline (TNP),2-(p-dimethyl-aminophenyl)ethenyl]-phenyl-methylene-propanenitrile(DPP-) andbis-[2-(p-dimethylaminophenyl)ethenyl]methylene-propanenitrile (BDP).Other up-converting phosphors, dyes and chelates are described in, butare not limited to, those described in Copending U.S. application Ser.No. 08/986,196 entitled "Production of Substantially MonodispersePhosphor Particles," filed on Dec. 5, 1997, Luminescent Materials,Ullmann's Encyclopedia of Industrial Chemistry, 5th Edition, Vol. A15,pgs. 519-557, and in Kirk-Othmer Encyclopedia of Chemical Technology,Third edition, Volume 14, pp. 527 ff, the disclosures of which areincorporated by reference in their entirety herein.

Luminescent labels of the invention include any material which emits orcan be induced to emit a signal. Typically, the luminescent labels maybe excited with radiation having a wavelength of from about 350 to about1500 nm. A signal emitted by the luminescent label is generally of awavelength in the infrared, visible or ultraviolet region. Also, asignal emitted by the luminescent label should be at a large enoughdown-converting or up-converting (Stokes or anti-Stokes) shift to enabledetection of the signal. Furthermore, the luminescent emissionpreferably does not overlap the emission of its matched up-convertinglabel unless time resolved detection methods are employed. Also, aluminescent label should not emit a signal at the same wavelength as theexcitation wavelength of its matched up-converting label.

Any luminescent labels used in conventional assays or, as describedabove, photophysical catalysis methods may be up-converting ordown-converting, phosphorescent or fluorescent materials. Theluminescent labels which are up-converting include, but are not limitedto up-converting phosphors, chelates, dyes and non-linear opticalcompounds, such as those described above. Luminescent labels which aredown-converting luminescent materials include, but are not limited to,down-converting phosphors, such as zinc sulfides activated with silverand those described in Copending U.S. application Ser. No. 08/986,196entitled "Production of Substantially Monodisperse Phosphor Particles,"filed on Dec. 5, 1997, Luminescent Materials, Ullmann's Encyclopedia ofIndustrial Chemistry, 5th Edition, Vol. A15, pgs. 519-557, and inKirk-Othmer Encyclopedia of Chemical Technology, Third Edition, Volume14, pp. 527 ff. the disclosures of which are incorporated by referencein their entirety. Luminescent label chelates include, but are notlimited to and those described in Hemmila et al. U.S. Pat. No.5,637,509, and Stavrianopoulos et al. U.S. Pat. No. 4,868,103, thedisclosures of which are herein incorporated by reference in theirentirety. Luminescent labels which are fluorescent dyes include, bit arenot limited to, TransFluoSpheres® (TFS) and those described in Mandel etal. U.S. Pat. No. 4,372,745, Brinkley et al. U.S. Pat. No. 5,326,692 andSinger et al. U.S. Pat. No. 5,573,909, the disclosures of which areherein incorporated by reference in their entirety. A preferredluminescent label is a down-converting fluorescent dye mixture soldunder the tradename TransFluoSpheres® (TFS) which is commerciallyavailable from Molecular Probes located in Eugene, Oreg. Generally,preferred luminescent labels of the invention have a high quantumefficiency and a high extinction coefficient.

Additionally, while down-converting and up-converting phosphor particlelabels of the invention may be prepared by conventional techniques andmethods known in the art, it is preferred that when particles such asup-converting phosphor particles are used, they should be substantiallymonodisperse particles. "Substantially monodisperse particles" areparticles which are substantially unagglomerated and, in general, arepredominately spherical in shape. In other words, the majority ofphosphorescent particles exist as individual particles in contrast toclusters of two or more phosphorescent particles. In a preferredembodiment, the substantially monodisperse phosphor particles are formedby a fluidized bed production process such as that described inCopending U.S. application Ser. No. 08/986,196 entitled "Production ofSubstantially Monodisperse Phosphor Particles," filed on Dec. 5, 1997.

The up-converting labels and luminescent labels may be attached to theirrespective probes by conventional methods, such as those described aboveor other methods known in the art. For example, an up-convertingphosphor label may be treated with a polymeric material to coat thephosphor to form a labeled probe. Another approach involves contactingan encapsulated luminescent having carboxyl groups on its surface withBiotin-LC-Hydrazide using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) under acidic conditions to form a stable peptide linkage betweenthe carboxyl groups on the latex sphere surface and the hydrazide of theBiotin-LC-Hydrazide. Biotin LC-Hydrazide and EDC are available fromPIERCE located in Rockford, Ill. Additional methods of attaching labelsto probes are described in Stavrianopoulos et al. U.S. Pat. No.4,868,103, and Tanke et al. U.S. Pat. No. 5,043,265, the disclosure ofwhich is incorporated by reference in its entirety.

With regards to the packaging of the compositions of the invention, thecompositions may be in the form of a kit comprising all of the essentialingredients required to conduct the desired assay or photophysicalcatalysis method. For example, the kit may contain matchingup-converting labeled probes and luminescent labeled probes. The assaykit is presented in a convenient, commercially packaged form. The kitcan be presented as a composition or as an admixture depending upon thecompatibility of the labeled probes. For example an assay kit can be apackaged combination having one or more containers, devices or the likeholding the labeled probes and other materials necessary for particularassay, and usually including written instructions for the performing theassay.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention inany manner.

EXPERIMENTAL EXAMPLES

Validation of Up-Converting Inorganic Phosphors as Reporters

Up-converting phosphor particles comprising sodium yttrium fluoridedoped with ytterbium-erbium were milled to submicron size, fractionatedby particle size, and coated with polycarboxylic acid. Na(Y₀.80 Yb₀.18Er₀.02)F₄ was chosen for its high efficiency upon excitation in therange 940 to 960 nm. A Nd:Yag pumped dye laser/IR dye combination wasused to generate 8-ns to 10-ns duration pulses in the above frequencyrange.

The laser pulses were used to illuminate a suspension of milled phosphorparticles in liquid and attached to glass slides in situ. The suspensionluminescence observed at right angles was monitored using a collectionlens, a spatial filter in order to filter out scattered excitation lightto the maximum possible extent, and a photomultiplier, vacuumphotodiode, or simple solid state photodiode (depending on the lightlevel observed).

The luminescent signal level was determined as a function of solution pH(range: 6-8), grain size, particle loading (μg/cm³), and the nature ofstabilizing anionic surfactant. Signals were recorded both as a timeintegral from a boxcar integrator and from a long RC time constant or asa transient signal using a transient digitizer in order to delineate theluminescence lifetime under particular experimental conditions. In situsignals were also measured by laser scanning microscopy. FIG. 11 is afluorescence scan of the phosphor emission spectrum incident toexcitation with a laser source at a wavelength maximum of 977.2 nm;emission maximum is about 541.0 nm. FIG. 12 is an excitation scan of thephosphor excitation spectrum, with emission collection window set at541.0 nm; excitation maximum for the phosphor at the 541.0 nm, emissionwavelength is approximately about 977 nm. FIG. 13 is a time-decaymeasurement of th e phosphor luminescence at 541.0 nm after terminationof excitation illumination; maximal phosphorescence appears atapproximately 400 μs with a gradual decay to a lower, stable level ofphosphorescence at about 1000 μs. FIG. 14 shows the phosphor emissionintensity as a function of excitation illumination intensity;phosphorescence intensity increases with excitation intensity up toalmost about 1000 W/cm³.

Phosphorescence efficiencies of submicron Na(Y₀.8 Yb₀.12 Er₀.08)F₄particles were measured. A Ti:sapphire laser was used as an excitationsource and a spectrophotometer and photomultiplier was used as adetection system. Two types of measurement were performed. The first wasa direct measurement in which the absolute emission per particle forphosphor suspensions was measured in emission bands at 540 nm and 660nm. The calibrated cross-sections are shown in FIG. 15, andsize-dependence is shown graphically in FIG. 16. This corresponded to aphosphorescence cross-section of approximately 1×10⁻¹⁶ cm² for 0.3 μmparticles with excitation light at 975 nm and an intensity ofapproximately 20 W/cm². The emission efficiency of dry phosphor powderof about 25 μm was also measured. On the basis of known values for theabsorption cross-section of Yb⁺³ in crystalline hosts (Lacovara et al.(1991) Op. Lett. 16:1089, incorporated herein by reference) and themeasured dependence of the phosphorescence emission on particle size, aphosphorescence cross-section of approximately 1×10⁻¹⁵ cm² was found.The difference between these two measurements may be due to a differencein phosphorescence efficiency between dry phosphor and aqueoussuspensions, or due to absorption of multiply scattered photons in thedry phosphor. On the basis of either of these cross-section estimates,the cross-section is sufficiently large to allow detection of singlesubmicron phosphor particles at moderate laser intensities. At laserintensities of roughly 10 W/cm², the phosphorescence scales as the laserintensity to the 1.5 power.

Phosphor Particle Performance: Sensitivity of Detection

A series of Terasaki plates containing serial dilutions of monodisperse0.3 μm up-converting phosphor particles consisting of (Y₀.86 Yb₀.08Er₀.06)₂ O₂ S were tested for up-conversion fluorescence under IR diodelaser illumination in a prototype instrument.

The phosphor particles were prepared by settling in DMSO and wereserially diluted into a 0.1% aqueous gum arabic solution. This appearedto completely eliminate any water dispersion problems. The serialdilutions used are listed in Table III.

                  TABLE III                                                       ______________________________________                                             Phosphor     Phosphor        Equivalent                                       Loading      Loading         Detection                                   Label                                                                              (ng/well)    (particles/well)                                                                              Sensitivity (M)                             ______________________________________                                        10.sup.0                                                                           1700 ± 90 23,6000,000 ± 1,200,000                                                                    4 × 10.sup.-12                        10.sup.-1                                                                          170 ± 9   2,360,000 ± 120,000                                                                        4 × 10.sup.-13                        10.sup.-2                                                                           17 ± 0.9 236,000 ± 12,000                                                                           4 × 10.sup.-14                        10.sup.-3                                                                           1.7 ± 0.09                                                                             23,600 ± 1,200                                                                             4, 10.sup.-15                               10.sup.-4                                                                          0.170 ± 0.009                                                                            2,360 ± 120,000                                                                           4 × 10.sup.-16                        10.sup.-5                                                                           0.017 ± 0.0009                                                                         236 ± 12     4 × 10.sup.-17                        10.sup.-6                                                                           0.0017 ± 0.00009                                                                       23.6 ± 1.2   4 × 10.sup.-18                        ______________________________________                                    

The stock DMSO dispersion had a phosphor density of 1.70±0.09 mg/mL (at95% confidence limits), determined gravimetrically by evaporating 4-1 mLsamples. This translates to 23.6×10⁹ particles/mL (assuming an averageparticle size of 0.3 μm and particle density of 5.3 g/mL). The residueafter evaporating the samples over the weekend at 110°-120° C. wasnoticeably yellow, but did phosphoresce when tested with an IR diodelaser.

Visual green light emanated from all serial dilutions down to 10⁻³(i.e., 1.7 μg/mL or 23.6×10⁶ particles/mL) in a 1 mLpolypropylenemicrofuge tube using a hand-held diode laser in a darkroom. The 10⁻¹ and 10⁻² dilutions were visibly cloudy. Either 1 μl ofeach serial dilution, or 0.1 μl of the next higher dilution, werepipetted into a well on the Terisaki plate. It was found that 1 μl fillsthe bottom of the well and 0.1 mu 1 spreads along the edge of the well,but does not cover the entire surface. Because of the statistical andpipetting problems associated with small volumes with low particleconcentrations, 2 to 4 replicates were prepared of each dilution.

The well of a Terasaki plate holds a 10 μl sample volume. Assuming allthe phosphor particles contained in this volume adhere to the bottom ofthe sample well, we can estimate an equivalent detection sensitivity(Table III). It should be noted that 10⁻¹⁵ to 10⁻¹⁸ M is the normalrange of enzyme-linked surface assays.

Control Sample Results

The control samples were scanned using a prototype up-conversionfluorimeter device (David Samnoff Research Center). The samples werescanned by moving the plate in 50 μm increments, using a motorized X-Ypositioning stage, relative to the focal point of an infrared diodelaser.

The IR diode laser was operated at 63 mW (100 mA). The beam was focusedto 2.4×10⁻³ cm² at the focal point. As the bottom of the sample well isabout 1.4×10⁻² cm² (1365 μm diameter), the beam covers less than 17% ofthe well bottom surface at any individual position. The well also hassloping side walls which widen from bottom to top of the sample well andare also interrogated by a progressively divergent laser beam.Neglecting losses in the optics, the IR light intensity at the focalpoint (bottom of the sample well) was approximately 26-27 W/cm² at 980nm wavelength. A photomultipler tube (PMT) was used for detection of thevisible (upconverted) light emitted from the sample. Since the laserbeam width was smaller than the surface area at the bottom of the samplewell, the plate was aligned by visual inspection against the focal pointof the diode laser so that the laser was centered in the middle well (C6when reading wells C5, C6 and C7, and D6 when reading wells D5, D6, andD7).

The PMT signal (amps) was recorded at each plate position andnumerically integrated over the width of the sample well (approximately4000 μm). Several scans were made at different positions in the 10⁻² to10° dilution sample wells to determine the uniformity of the particledistribution. The background signal was determined by integrating theaverage dark field current of the PMT over a 4000 μm distance, whichyields an integrated background signal of 1×10⁻⁹ μa-m. The integrationproducts of the samples wells were scaled to this background signal, andare shown in FIG. 19.

Immimodiagnostic Sample Detection

A series of IgG/anti-IgG samples for demonstrating the capabilities ofthe up-converting phosphor reporters in a immunosorbant assay format wasprepared. These samples consisted of six individual wells (positivesamples) coated with antigen (mouse IgG) and bovine serum albumin (BSA),and six wells coated with BSA alone (negative controls). Nominal 0.3 μm(Y₀.86 Yb₀.08 Er₀.06)₂ O₂ S phosphor particles coated with goatanti-mouse IgG antibody (anti-IgG) were then used as thereporter-antibody conjugate. Six wells (C5, C6, C7, D5, D6, and D7) of aclear polystyrene Terasaki plate were coated with mouse IgG byincubating at 37° C., against 5 μL of a 100 μg/μL mouse IgG solution inphosphate buffered saline (PBS). After 1 h, this solution was aspiratedoff and each sample well was washed with 10 μL of 3% BSA in PBS. Thiswas immediately aspirated off and replaced with 20 μL of 3% BSA in PBS.Each sample well was post-coated with BSA by incubating against the 20μL of BSA/PBS solution for 1 h at 37° C. The post-coat solution wasaspirated off and the plates stored at 4° C. overnight. These wells wereconsidered in positive samples. The same six wells in a second Terasakiplate were prepared in an identical fashion, except they were not coatedwith mouse IgG. This second set of sample wells were considered negativecontrols.

Phosphor-Antibody Conjugate

A solution of (Y₀.86 Yb₀.08 Er₀.06)₂ O₂ S phosphor particles wasprepared by suspending the dry phosphors into DMSO. The initial particledensity was approximately 10⁷ particles/mL as determined by counting thenumber of particles contained in the field of an optical microscope. Itshould be noted that the 0.3 μm fundamental particle size was below theresolution limits of the microscope. This solution was allowed to settleundisturbed for 3 days. The supernatant, which was turbid and presumablycontained mostly monodisperse smaller particles was used for subsequentconjugation.

Goat anti-mouse IgG antibody (Ab) was conjugated (by adsorption) ontothe DMSO fractionated phosphor particles. This was done by mixing 200 μLof the Absolution (in 0.1M Tris-HCl, pH 7.2) with 100 μL of the phosphorsuspension in DMSO. Several different Ab concentrations were tried inthe range of 0.025 to 1 μg/μL. A concentration of 0.25 μg/μL appeared toresult in the most efficient coating (i.e., maximum Ab utilization witha minimum of clumping of the phosphor particles). The phosphors wereequilibrated overnight at room temperature with the Ab in this DMSO/Trissolution with gentle agitation. The resulting phosphor-Ab conjugateswere centrifuged from this solution and resuspended in a 3 μg/μL BSAsolution in PBS for post-coating. The resulting BSA/PSA resuspension wasused directly for the assay.

The degree of Ab adsorption to the phosphors, and residual Ab activity,was determined by titrating the phosphor-bound Ab with a fluoresceinisothiocyanate (FITC) conjugated-mouse IgG. The resulting FITC-labeledphosphors were passed through a Cyteron Absolute flow cytometer, whichwas also capable of measuring the relative size of the particles. Twodistinct size subpopulations were observed with about 65% of the countedparticles appearing as small, presumably monodisperse particles, and 35%being significantly larger, presumably aggregates. Only 60% of thesmaller subpopulation appeared to have significant quantities of activeAb (determined by FITC fluorescence). Of the purported aggregates, about90% appeared to contain active Ab (by FITC fluorescence). This suggeststhat less than 40% of the phosphor-Abconjugates were of an appropriatesize (nominal 0.3 μm) and exhibited anti-mouse IgG activity. A similarfraction of phosphor-Ab conjugates (31%) were active but carried asignificantly larger phosphor reporter.

The PMT signal (amps) was recorded at each plate position andnumerically integrated over the width of the sample well approximately4000 μm). The average signals (with 95% confidence limits) are:

Average of Positive Samples=1.30×10⁻⁴ ±1.25×10⁻⁴ μa-m

Average of Negative Controls=4.20×10⁻⁶ ±6.82×10⁻⁶ μa-m

The positive samples and negative controls are statistically differentat the 99.9% confidence level. The positive samples emit on average30.0±29.7 times more light than the negative controls.

Linkage of Phosphors to Biological Macromolecules

In order to delineate further the parameters for up-converting phosphorsas biochemical reporters, biological linkers were attached to phosphorparticles. Sodium yttrium fluoride-ytterbium/erbium phosphor particleswere coated with streptavidin. The excitation and emission spectralproperties of the phosphor alone and the phosphor coated withstreptavidin were measured (FIGS. 17A, 17B, 18A, and 18B) and both theuncoated and streptavidin-coated phosphors were almost identical intheir absorption and emission properties, indicating that the attachmentof macromolecular linkers (e.g., proteins) have little if any effect onthe phosphorescent properties of the up-converting phosphor. Thestreptavidin-coated phosphors were then specifically bound tobiotinylated magnetic beads, demonstrating the applicability oflinker-conjugated inorganic phosphors as reporters in biochemicalassays, such as immunoassays, immunohistochemistry, nucleic acidhybridizations, and other assays. Magnetic bead technology allows forthe easy separation of biotin-bound streptavidin-coated phosphor from asolution, and is particularly well-suited for sandwich assays whereinthe magnetic bead is the solid substrate.

Advantageously, streptavidin-biotin chemistry is widely used in avariety of biological assays, for which up-converting phosphor reportersare suited. FIG. 20 shows schematically, for example and not limitation,one embodiment of an immunoassay for detecting an analyte in a solutionby binding the analyte (e.g., an antigen target) to a biotinylatedantibody, wherein the analyte forms a sandwich complex immobilized on asolid substrate (e.g., a magnetic bead) by linking a first bindingcomponent bound directly to the solid substrate to a second bindingcomponent (e.g., the biotinylated antibody); a streptavidin-coatedup-converting phosphor then binds specifically to the biotinylatedantibody in the sandwich and serves to report formation of the sandwichcomplex on the solid substrate (which is a measure of the analyteconcmagnetic bead., it is solid substrate is a magnetic bead, it isreadily removed from the sample solution by magnetic separation and theamount of phosphor attached to the bead(s) in sandwich complex(es) aredetermined by measuring specific up-converting phosphorescence. Thus,sandwich complex phosphorescence provides a quantitative measure ofanalyte concentration.

Biotinylated polynucleotides are also conveniently used as hybridizationprobes, which can be bound by streptavidin-coated up-convertingphosphors to report hybrid formation.

Background Phosphorescence in Biological Samples

Background signals were determined in two biological samples fordetermination of potential background in immunoassays. Sputum and urinewere used as samples in the same apparatus as used for thephosphorescence sensitivity measurements (supra). No background levelswere found above the system noise levels set by the photomultiplier darkcurrent. This noise level allows detection of signals from on the orderof a few hundred particles cm³.

This is close to a single particle in the detection volume of thesystem.

A photomultiplier is a preferred choice for a detector for highsensitivity measurements of up-converting phosphors sincephotomultipliers can be selected to produce high quantum efficiency atthe up-converted (i.e., emitted) wavelengths and virtually no responsein the range of the longer excitation wavelengths.

Detection of Cell Antigens with Phosphor-Labeled Antibodies

Streptavidin is attached to the up-converting phosphor particles asdescribed, supra. The mouse lymphoma cell line, EL-4, is probed with ahamster anti-CD3 antibody which specifically bind, to the 30 kD cellsurface EL-4 CD3 Tlymphocyte differentiation antigen. The primaryhamster antibody is then specifically bound by a biotinylatedgoat-antihamster secondary antibody. The biotinylated secondary antibodyis then detected with the streptavidin-phosphor conjugate. This type ofmultiple antibody attachment and labeling is termed antibody layering.

Addition of multiple layers (e.g., binding the primary hamster Ab with agoat-antihamster Ab, followed by binding with a biotinylatedrabbit-antigoat Ab) are used to increase the distance separating thephosphor from the target. The layering effect on signal intensity andtarget detection specificity is calibrated and optimized for theindividual application by performing layer antibody layering from onelayer (primary antibody is biotinylated) to at least five layers andascertaining the optimal number of layers for detecting CD3 on EL-4cells.

FIG. 21 schematically portrays simultaneous detection of two EL-4 cellsurface antigens using phosphors which can be distinguished on the basisof excitation and/or emission spectra. Detection of both antigens in thescheme shown in FIG. 21 uses a biotinylated terminal antibody which isconjugated to streptavidin-coated phosphor (#1 or #2) prior toincubation with the Ab-layered sample. Thus, the phosphor-antibodyspecificity is retained through the unusually strong (K_(D) approx.1×10¹⁵ M⁻¹) non-covalent bond between streptavidin and biotin which ispre-formed before incubation with the primary antibody-bound sample.Quantitation of each antigen is accomplished by detecting the distinctsignal(s) attributable to each individual phosphor species.Phosphorescent signals can be distinguished on the basis of excitationspectrum, emission spectrum, fluorescence decay time, or a combinationof these or other properties. FIG. 22 shows a schematic of an apparatusfor phase-sensitive detection, which affords additional backgrounddiscrimination. The pulse or frequency mixer is set to pass the signaland discriminate against the background following frequency calibrationfor maximum background rejection.

Covalent Conjugation of Upconverting Phosphor Label to Avidin

An upconverting ytrium-ytterbium-erbium (Y₀.86 Yb₀.08 Er₀.06) oxysulfide(O₂ S) phosphor was linked to avidin by the following procedure:

Monodisperse upconverting phosphor particles were silanized withthiopropyltriethoxysilane (Huls) following the procedure detailed byArkles (in: Silicone Compounds: Register and Review, Hills America, pgs.59-75, 1991).

This consisted of adding thiopropyltriethoxysilane (2 g) and 95% aq.ethanol (100 mL) to a 500 mL Erlenmeyer flask and stirred for 2 minutes.Approximately 8 mL of the 65 mg/mL phosphor suspension in DMSO was thenadded to the mixture. This suspension was stirred for an additional 2minutes, then transferred to centrifuge tubes and centrifuged toseparate the phosphor particles. The pellets were washed twice with 95%aq. ethanol centrifuging each time. The resulting particles werecollected and dried overnight under vacuum at approximately 30° C.Aquantity (127 mg) of dry silanized phosphors were resuspended in 1.5 mLof DMSO (phosphor stock).

A solution containing 1.19 mg of avidin (Pierce) in 1.0 mL of boratebuffer (954 mg sodium borate decahydrate and 17.7 mL of 0.1N NCl in 50mL of deionized water, pH 8.3) was prepared (Avidin stock). Anothersolution containing 1.7 mg of N-succinimide(4-iodoacetyl) aminobenzoate(Pierce Chemical) in 1.2 mL of DMSO was prepared (SIAB stock). Aquantity (10 mu L) of the SIAB stock was added to the 1.0 mL of Avidinstock and stirred at room temperature 30 min to allow theN-hydroxysuccimide ester of the SLAB to react with primary amines on theavidin (Avidin-SIAB stock).

A 20 mL scintillation vial was prepared containing 10 mL of boratebuffer (pH 8.3). The following additions were then made to this vial:21.6 μL of the avidin-SIAB stock solution followed by 1.5 mL of thephosphor stock. This reaction mixture was stirred at room temperature inthe dark overnight to allow the SIAB activated avidin to react with thethiol groups present on the silanized phosphor surface and resulting inthe covalent linkage of avidin to the phosphor particles.

After the overnight incubation 1.0 mL of the reaction mixture wascentrifuged (1 min at 10,000 g) and the supernatant removed. The pelletwas resuspended in 1.0 mL of phosphate buffered saline (pH 7.2, Pierce)and centrifuged again to wash any uncongugated protein from thephosphors. This washing process was repeated. The washed pellet wasresuspended in 1.0 mL of phosphate buffered saline and used directly indiagnostic assays as described below.

Measurement Apparatus

A modified SLM Aminco 48000 Fluorimeter was used to measure thefluorescence spectrum from the phosphor samples. The modifications tothis device consisted of adding a laser diode (David Sarnoff CD-299R-FA#13) which was input to the fluorimeter through port 3. The laser diodeemits at λ=985.1 nm. Spectral data provided by the David SarnoffResearch Center also shows a small peak at 980.2 nm. This peak has 15%the intensity of the peak at 985 nm. A 5.08 cm focal length lens wasused to collimate the diode laser beam. The power of the IR laser lightwas measured as 6.1 mW at the cuvette location with a drive current of75 mA. The beam was not focused at the center of the cuvette. This istrue for the standard visible light from the fluorimeter excitationmonochromator as well. The laser diode beam is diverging as it entersthe cuvette holder and is approximately 4 mm (H)×2 mm (V) by the time itreaches the center of the cell, neglecting the changes in refractiveindex of the cell wall and the liquid.

Light emitted is scanned with a monochromator and detected by aphotomultiplying tube (PMT) 90° from the direction of the excitationlight. The detection limits for the modified SLM Aminco 48000 weredetermined by serial dilution to be 4×10⁻¹⁶ M (240,000 phosphorparticles per mL) in PBS. Phosphor emission peaks in the spectrum wereseen at wavelengths of 406±2 nm, 434±2 nm, 522±2 nm, and 548±2 nm. Thelargest peak was at 548 nm. The intensity of the 548 nm peak was used todiscriminate samples.

Linkage of Avidin-Phosphor Conjugate to Cell Surface Marker

A lymphoblastoid cell line (Human Genetic Mutant Cell Repository#GM07092) was cultured in RPMI 1640 media containing 15% heatinactivated fetal calf serum. A suspension of cells (10⁷ cells) wascentrifuged and resuspended in an equal volume of phosphate bufferedsalin (PBS) pH 7.4. Cells were washed two times in PBS and resuspendedto a final concentration of 5×10⁶ cells/ml. These cells were thenincubated with a mouse IgG1 monoclonal antibody to human β₂microglobulin, a Class I histocompatibility antigen in polystyrenecentrifuge tubes. The cells were immunoprecipitated for 30 minutes at 4°C. with an antibody concentration of 10 μg/ml. The cells were harvestedby centrifugation, washed twice in PBS, resuspended in PBS and thenaliquoted (250 μL) into six fresh centrifuge tubes. Four of thesesamples received biotinylated goat anti-mouse IgG, while the remainingtwo received FITC.-labelled goat anti-mouse IgG. Theseimmunoprecipitations were performed at 4° C. for 30 minutes in volume of400 μL with a final second antibody concentration of 20 mu g/ml. Thecells were harvested and washed in PBS as above but were resuspended in50 μL of blocking buffer (0.2% purified casein in PBS, Tropix, Bedford,Mass.). The cell-antibody complexes were blocked in this solution for 30minutes at room temperature and then transferred to fresh tubes.

A pre-blocked suspension (40 μL) of either avidin-Phosphor conjugate,avidin-FITC, avidin, or unconjugated Phosphor was added to four of thecell samples conjugated with the biotinylated anti-mouse IgG (H&L). Inaddition, an equal amount of pre-blocked avidin-Phosphorer unconjugatedPhosphor was added to the remaining two cell samples immunoprecipitatedwith the non-biotinylated FITC-labelled anti-mouse IgG (H&L). The avidinreporter conjugates or negative controls were pre-blocked as follows.Avidin-Phosphor and Phosphor alone was diluted in blocking buffer byadding 10 μL of a 6.7 mg/ml suspension to a final volume of 100 μL.Avidin-FITC and the avidin alone controls were also diluted in blockingbuffer by adding 27 μL of 2.5 mg/ml solution to a final volume of 100μL. These reagents were blocked at room temperature for 3 hours withintermittent resuspension and then added to 50 μL of cells labelled withbiotinylated or non-biotinylated second antibody. The avidin-biotinreactions were performed at room temperature for 30 minutes withoccasional resuspension. The reactions were stopped by harvesting thecells by centrifugation and washing twice in blocking buffer. Thesamples were resuspended in 100 μL of blocking buffer and allowed tosettle for 4-5 minutes. Slides for imaging were prepared by pipetting 5μL of settled cells from the bottom of the tube. Cells were imaged byconfocal laser microscopy under appropriate conditions to observe cellsurface FITC and upconverting phosphor signals. The observations aresummarized in Table IV.

                  TABLE IV                                                        ______________________________________                                                                      Cell    Cell                                                                  Surface Surface                                      Type of goat                                                                              Avidin       Phosphor                                                                              FITC                                    Tube anti-mouse IgG                                                                            Conjugate    Signal  Signal                                  ______________________________________                                        1    biotinylated                                                                              Avidin-Phosphor                                                                            +       -                                       2    biotinylated                                                                              Phosphor     -       -                                       3    biotinylated                                                                              Avidin       -       -                                       4    biotinylated                                                                              Avidin-FITC  -       +                                       5    FITC labelled                                                                             Avidin-Phosphor                                                                            -       +                                       6    FITC labelled                                                                             Phosphor     -       +                                       ______________________________________                                    

The remainder of the samples were used to resuspended paramagnetic,polystyrene beads bound with sheep anti-mouse IgG. For each of the sixsamples, 3×10⁷ beads were prewashed with blocking buffer for 1 hour atroom temperature in Eppendorf tubes. The buffer was removed byaspiration while the tubes were in a magnetic rack. The magnetic beadswith anti-mouse IgG were allowed to bind to the antibody labelled cellsfor 1 hour at room temperature with intermittent resuspension. Themagnetic beads were then collected on a magnetic rack, washed four timesin blocking buffer, resuspended in 100 μL blocking buffer, transferredto a fresh tube, and up-converting phosphorescence was measured on thefluorimeter.

To scan for phosphor emission, the emission monochromator bandwidth wasset to 8 nm and the spectra were scanned from 500 to 700 nm with a stepsize of 2 nm. Samples were also measured for FITC signal by exciting thesamples with 37 μM at λ=490 nm with a 2 nm bandwidth. Since theexcitation wavelength (490 nm) and the emission wavelength (514 nm) arevery close for FITC, higher resolution was required to get separablesignals than with phosphor labelling. The intensity of the 490 nm signalwas 240 μW/cm² at the center of the well. FITC emission spectra werescanned at 0.5 nm increments from 450 nm to 750 nm with a 2 nm bandwidthon the emission monochromator. Sample 1 is the positive control andclearly yielded the highest emission signal. Sample 2 indicates that anynonspecific adsorption of the phosphors to the sample is limited and isreadily discriminated from signal attributable to avidin-conjugatedphosphor and showing that avidin linked phosphors can specifically bindonly when they are conjugated with the probe, in this example throughthe biotin-avidin linkage. Sample 3 is the negative control whichcontains no phosphors, only avidin. Sample 4 shows FITC-conjugatedavidin. Although FITC signals were observed on the cell surface by lasermicroscopy, the signals were below the level of detection on thefluorimeter for measurement of FITC, and since there was no phosphor inthe sample there was no significant phosphor signal. Samples 5 and showthat FITC-conjugated primary antibodies can be detected and that thepresence of phosphor or avidin-phosphor does not significantly disruptbinding of the primary antibody to its target antigen.

Linkage of Avidin-Phosphor Conjugate to DNA

Plasmid DNA (25 μg) was nick translated in the presence of 20 mM dGTP,20 mM dCTP, 20 mM biotin-14 DATP, 13 mM dTTP, and 7 mM digoxigenin-11dUTP and purified by ethanol precipitation. The average size of thebiotinylated, digoxygenin labelled fragments was estimated to be between200-300 nucleotides as estimated by gel electrophoresis. Approximately20 μg DNA was immunoprecipitated for 1 hour at 22° C. with 10 μg/mlmouse monoclonal anti-digoxigenin IgG1 solution (PBS) in a 200 μLvolume. An equivalent reaction containing no DNA was also prepared. Eachof the two samples were then aliquoted (50 μL) into three freshEppendorf tubes.

The avidin-conjugates were blocked for 1 hour at room temperature bydiluting 500 μg of an avidin-phosphor suspension, unconjugated phosphorsuspension, or avidin solution in 300 μL of blocking buffer. For each ofthe samples (summarized below in Table V) 50 μL of the anti-digoxigeninconjugates was added to 150 μL of pre-blocked avidin-conjugates oravidin and were incubated for 30 minutes at room temperature.

Unbound avidin-conjugates were removed by resuspended 3×10⁷ paramagneticbeads linked with sheep anti-mouse IgG (pre-blocked in blocking buffer).After incubation for 30 minutes at room temperature with intermittentresuspension, the beads were separated on a magnetic rack and washed 4to 6 times in PBS. The antibody-DNA bound beads were then measured onthe fluorimeter.

The samples were scanned from 500 to 700 nm with a bandwidth of 8 nm andstep size of 2 nm. Each PMT value reported (Table V) represents anaverage over 5 scans. Sample 1 is expected to provide the highest PMTsignal since biotinylated DNA is present and can bind to theavidin-linked phosphors. Sample 2 indicates the level of nonspecificadsorption of the phosphors to the sample which is found to beinsignificant since the PMT signal is observed to be the same as that ofthe negative control (sample 4) which contains no phosphors. Sample 3 isanother control and shows that the avidin-linked phosphors do not bindto the paramagnetic beads in the absence of DNA. Samples 5 and 6 showresults of FITC-labeled avidin used to validate assay.

                  TABLE V                                                         ______________________________________                                        Upconverting Phosphor Nucleic Acid Diagnostic Assay Results                   Sam-                       PMT Signal                                                                             PMT Signal                                ple  DNA         Reporter  (V @ 546 nm)                                                                           (V @ 514 nm)                              ______________________________________                                        1    DNA labeled with                                                                          Avidin    6.1297   2.4788                                         digoxigenin and                                                                           linked                                                            biotin      Phosphor                                                     2    DNA labeled with                                                                          Silanized 1.0528   4.4022                                         digoxigenin and                                                                           Phosphor                                                          biotin                                                                   3    No DNA      Avidin    1.6302   3.5779                                                     linked                                                                        Phosphor                                                     4    DNA labelled                                                                              Avidin    0.8505   2.8067                                         with digoxigenie                                                              and biotin                                                               5    DNA labelled                                                                              FITC-Avidin                                                                             1.0484   8.4394                                         with digoxigenin                                                         6    No DNA      FITC-Avidin                                                                             1.0899   3.5779                                    ______________________________________                                    

Phosphor Downconversion Evaluation

A sample of the (Y₀.86 Yb₀.08 Er₀.06)₂ O₂ S phosphors were scanned forthe presence of a downconverted signal. This was accomplished byexciting a sample of the monodisperse phosohors described above (4×10⁻¹²M in DMSO) with 1.3 mW of monochromatic light at 350 nm with a 6 nmbandwidth for the excitation source. Detection was accomplished byscanning this sample from 350 to 800 nm with a monochomator bandwidth of8 nm. Scanning was performed in 2 nm increments. No downconversion wasobserved. Moreover, no downconversion was seen at the excitationwavelengths cited by Tanke et al. (U.S. Pat. No. 5,043,265) Thus, theupconverting phosphors tested are unlike those reported in Tanke et al.

HOMOGENEOUS ASSAYS

The multiphoton activation process characteristic of upconvertingphosphors can be exploited to produce assays that require no samplewashing steps. Such diagnostic assays that do not require the removal ofunbound phosphor labels from the sample are herein termed homogeneousassays, and can also be termed pseudohomogeneous assays.

Homogeneous Assay Example 1

One embodiment of a homogeneous assay consists of the use of anupconverting phosphor label linked to an appropriate probe (e.g., anantibody or DNA). The phosphor-labeled probe specifically binds to atarget (e.g., antigen or nucleic acid) that is linked to a capturingsurface. A suitable capture surface can be the tip of a light carryingoptical fiber (FIG. 23) or the bottom surface of a sample container(FIG. 24). Upon incubation of the target-labelled capture surface withthe phosphor-labelled probe, phosphor particles will accumulate at thecapture surface as a function of the amount of target present on thecapturing surface. The target may be linked directly to the capturingsurface or may be immobilized by interaction with a binding agent (e.g.,specific antibody reactive with target, polynucleotide that bindstarget) that is itself linked to the capturing surface (such as in asandwich immunoassay, for example).

Detection of the phosphor bound to the capture surface is effected usingan excitation light that is focused from a low intensity beam of largecross-section to a high intensity beam of small c:ross-section with thefocal point of the beam being an or very near the capture surface.Focusing of the excitation light is accomplished by transmission throughoptical elements that have a very small focal length, such that the beamdiverges and becomes less intense, within a short distance of thecapture surface.

Since the intensity of the light emitted from the upconverting phosphorlabels is proportional to the excitation light intensity raised to apower of two or greater, phosphors near the focal point of theexcitation source will emit significantly more light than thoseremaining in suspension in the sample away from the capture surface.Therefore, binding of upconverting phosphor linked probes to the capturesurface will yield an increase in emitted light intensity measured fromthe sample as a whole or as measured from a control sample in whichphosphors do not bind to the capture surface. Emitted light intensitymay be plotted as a function of target concentration using forstandardization (calibration) a series of samples containingpredetermined concentrations of target. The emitted light intensity froma test sample (unknown concentration of target) can be compared to thestandard curve thus generated to determine the concentration of target.

Examples of suitable homogeneous assay formats include, hut are notlimited to, immunodiagnostic sandwich assays and antigen and/or antibodysurface competition assays.

Homogeneous Assay Example 2

Another embodiment allows for the accumulation of upconverting phosphorlinked probes at the detection surface by the application of centrifugalor gravitational settling. In this embodiment an upconverting phosphoris linked to multiple probes. All the probes must bind to the sametarget, although said binding can be accomplished at different locations(e.g., as antibody probes may target different epitopes on a singleantigen). The multiprobe phosphor can then be used to effect theaggregation of targets in solution or suspension in the sample. Thisaggregation will result in the formation of a large insolublephosphor-probe-target complex that precipitates from solution orsuspension (FIG. 25). The aggregated complex containing phosphorsaccumulates at a detection surface while nonaggregated material remainsin solution or suspension. Detection is accomplished as described in theabove example using a sharply converging excitation beam.

Evaluation of Up-converting Chelate

Up-conversion has been performed in rare earth chelate and rare earthsalt solutions. Chelate of erbium and neodymium have been prepared withethylenediaminetetraacetic acid (EDNA) and dipicolinic acid (DPA). Theerbium chelate were pumped using light near 793.5 nm from a Ti:sapphirelaser (the excitation scheme of Macfarlane (1989) Appl. Phys. Lett.54:2301). This approach produced upconversion but not satisfactorily,which we attribute to weak absorption for the first step due to theincrease in linewidth in the chelate over the low temperature crystalused for the up-conversion laser.

The neodymium chelate were excited with light near 580 nm from aNd:YAG-pumped dye laser (following the excitation scheme of Macfarlaneet al. (1988) Appl. Phys. Lett. 52:1300). An emission spectrum for theemitted up-converted light at 380 nm is shown in FIG. 32. We estimatethe up-conversion cross section to be 10₋₂₇ cm² for this experiment.

We have also observed up-conversion in thulium acetate hexahydrate andholmium chloride hexahydrate in solution following the excitationschemes of Allain et al. (1990) Electron. Lett. 26:166, and Allain etal. (1990) Electron. Lett. 26:261, respectively. The salts weredissolved in heavy water, and excitation was performed using a kryptonlaser. Although the up-conversion was weak, the up-conversion should beimproved if chelated compounds are used instead of dissolved salts.

MATCHING LABEL EXAMPLES

Matching Label Example 1

A study was made of the excitation of an up-converting label where itssubsequent energy emission excites a luminescent label causing theluminescent label to emit radiation at a wavelength which can bedetected. Specifically, the secondary fluorescent emission of aninfrared excited up-converting phosphor label coupled with a luminescentlabel was studied.

The up-converting label, a phosphor, (Y,Yb, Tm)₂ O₂ S phosphor having anemission radiation of 488 nm, was dispersed in water by sonification andthen diluted to 0.1 mg/ml in water. The luminescent label,TransFluoSpheres ® (TFS) having an excitation wavelength of 488 nm and aparticle diameter of 0.35 microns, was dispersed in water bysonification and diluted in water to 0.2% solids. Ten microliters of thephosphor and ten microliters of TFS were then mixed and dried on a glassslide at 60° C. and then mounted on a coverslip. The glass slide wasthen subjected to excitation radiation having a wavelength of 488 nmwhich produced a very bright TFS fluorescence against a mottled greenbackground. The glass slide was also subjected to an excitationradiation having a wavelength of 900 to 1000 nm. A faint bluewhiteluminescence characteristic of the phosphor was detected by visualobservation as well as areas of dim yellow/green fluorescence. Theyellow/green fluorescence represented the presence of TFS:phosphoraggregates in which the TFS was excited by the phosphor emissionradiation.

Matching of Example 2

The attachment of a luminescent label to a probe was studied.Specifically, the biotinylation of a secondary luminescent latexparticle with a linker arm containing a biotin-complex was studied. Thesecondary luminescent studied was a TransFluoSpheres ® (TFS) latexparticle available from Molecular Probes of Eugene, Oregon, having anexcitation radiation of 488 nm, a particle diameter of 0.35 microns anda carboxylate modified surface (free --COOH groups exposed).

The TFS particle was reacted with EZ-link Biotin-LC-Hydrazide using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) suspended at 0.2%solids in phosphate buffered saline. An avidin horseradish peroxidaseconjugate (Avidin-HRP) was reconstituted in water to 1 mg/ml. To fixedvolumes of the TFS-biotin various concentrations of Avidin-HRP, 0.1 to100 micrograms/ml were added. The TFS-biotin/Avidin-HRP mixture was thenincubated at room temperature for 30 minutes after which the mixture wascentrifuged and aspirated to form a dry pellet. The dry pellet wassuspended in 100 microliters of Biotin-LC-Hydrazide and incubated for 30minutes in the dark.

The mixture was observed for changes in color as an indication of HRPactivity due to coupling of the Avidin-HRP to TFS-Biotin. An intenseblue color was observed at all concentrations of Avidin-HRP.

Matching Label Example 3

The conjugation of an up-converting label to a probe was studied.

One milliliter of a 0.1 mg/ml up-converting phosphor label solution wascentrifuged for ten minutes. The supernatant was then removed and 3 mlof Hepes buffer (pH 6.3) was added. The up-converting phosphor particleswere then sonicated until the particles dispersed. Another 7 ml of Hepesbuffer was added to the dispersion. The dispersion was then centrifugedfor ten minutes. The supernatant was then removed. Three milliliters ofa 0.1 1 mg/ml solution of NeutrAvidin in Hepes buffer, available fromPIERCE of Rockford, Ill., was then added to the up-converting phosphorparticles. This mixture was then stirred for two hours in the dark atroom temperature and then centrifuged. The supernatant was removed and10 ml of Hepes buffer was added without dispersing the particles. Theparticles were then centrifuged. The step of removing the supernatant,adding Hepes buffer without dispersing the particles and centrifugingthe particles was then repeated an additional two times. This wasfollowed by twice adding 3 ml of Hepes buffer to the particles,sonicating the particles until they dispersed, adding an additional 7 mlHepes buffer, centrifuging the particles and then removing thesupernatant. Then 3 ml of a Hepes buffer having a pH of 8.5 andcontaining 0.1% bovine serum albumin was added to the particles. Theparticles were then sonicated until dispersed and an additional 7 ml ofthe Hepes pH 8.5 buffer was added. The particles were then centrifugedand the supernatant was removed. Then the particles were avidinylated in2 ml of Hepes buffer having a pH of 8.5 and containing 0.1% bovine serumalbumin.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

The claimed invention is:
 1. A composition for detecting an analyte in asample using matched labels, comprising:a pair of matched up-convertingand luminescent labeled probes, wherein the up-converting labeled probeis an inorganic phosphor particle comprising at least one rare earthelement and a phosphor host material, and the luminescent label ismatched to the up-converting label.
 2. The composition according toclaim 1, wherein the probes are antibodies, polynucleotides, polypeptidehormones, receptor ligands, streptavidins, Staphylococcus aureus ProteinA, lectins, antigens or mixtures thereof.
 3. The composition accordingto claim 1, wherein the target analyte is a polynucleotide, apolypeptide, a virus, a microorganism, a hapten, a mammalian cell, ahormone, a glycoprotein, a blood clotting factor, a lipoprotein, a drug,a polysaccharide, a metabolite, a pollutant, a pesticide or mixturesthereof.
 4. The composition according to claim 1, wherein thecomposition is in the form of an assay kit.
 5. The composition accordingto claim 1, wherein the up-converting label is an up-convertinginorganic phosphor and comprises at least one rare earth element and theprobes are antibodies, polynucleotides, polypeptide hormones, receptorligands, streptavidins, Staphylococcus aureus Protein A, lectins,antigens or mixtures thereof.
 6. A method for detecting an analyte in asample with the composition of claim 1 using matched labels, comprisingthe steps of:contacting a sample suspected of containing a targetanalyte with matched up-converting and luminescent labeled probe pairs,wherein the up-converting labeled probe and the luminescent labeledprobes are capable of binding the target analyte to form a labeledprobe-target complex; illuminating the sample to excite a label of thelabeled probe-target complex; and observing the sample for emissionradiation from the matched label of the labeled probe-target complex,thereby detecting the presence or absence of the analyte.
 7. A methodaccording to claim 6, wherein the up-converting label is an excitationlabel and the luminescent label is an emission label.
 8. A methodaccording to claim 7, wherein the illumination step excites theexcitation label with radiation having a wavelength ranging from about700 to about 1500 nm.
 9. A method according to claim 6, wherein theluminescent label is an excitation label and the up-converting label isan emission label.
 10. A method according to claim 9, wherein theillumination step excites the excitation label with radiation having awavelength ranging from about 350 to about 1500 nm.
 11. A methodaccording to claim 10, wherein the illumination step excites theexcitation label with radiation having a wavelength ranging from about350 to about 700 nm.
 12. A method according to claim 6, wherein thesample is contacted with two or more matched labeled probe pairs, thematched labeled probe pairs having matched labels with differentexcitation label wavelengths and different emission label wavelengths,and wherein the matched probe pairs bind to different target analytes.13. A method according to claim 6, wherein the sample is contacted withtwo or more matched labeled probe pairs, the matched labeled probe pairshaving matched labels with similar excitation label wavelengths anddifferent emission label wavelengths, and wherein the matched probepairs bind to different target analytes.
 14. A method according to claim6, wherein the sample is contacted with two or more matched labeledprobe pairs, the matched labeled probe pairs having matched labels withdifferent excitation label wavelengths and similar emission labelwavelengths, and wherein the matched probe pairs bind to differenttarget analytes.
 15. A method according to claim 6, wherein the sampleis contacted with two or more matched labeled probe pairs, the matchedlabeled probe pairs having matched labels with similar excitation andemission label wavelengths, and wherein the matched labels are attachedto probes which bind affinitively to different target analytes.
 16. Amethod according to claim 6, wherein the target analyte is apolynucleotide, a polypeptide, a virus, a microorganism, a hapten, amammalian cell, a hormone, a glycoprotein, a blood clotting factor, alipoprotein, a drug, a polysaccharide, a metabolite, a pollutant, apesticide and mixtures thereof.
 17. A method according to claim 6,wherein the probes are antibodies, polynucleotides, polypeptidehormones, receptor ligands, streptavidins, Staphylococcus aureus ProteinA, lectins, antigens and mixtures thereof.
 18. A method according toclaim 6, wherein one of the labeled probes is bound to a solid support.19. A method according to claim 6, further comprising before theillumination step, the step of separating the labeled-probe targetcomplex from any unbound labeled probes.